Immunoglobulin variants and uses thereof

ABSTRACT

The invention provides humanized and chimeric anti-CD20 antibodies for treatment of CD20 positive malignancies and autoimmune diseases.

This is a continuation application of U.S. patent application Ser. No.12/256,349 filed Oct. 22, 2008, which is a divisional of Ser. No.11/147,780, filed Jun. 7, 2005, U.S. Pat. No. 7,799,900 issued Sep. 21,2010, which is a continuation application of international patentapplication number PCT/US03/40426, filed Dec. 16, 2003, which claimsbenefit of provisional application Ser. No. 60/526,163, filed on Dec. 1,2003 and provisional application Ser. No. 60/434,115, filed on Dec. 16,2002, which applications are incorporated herein by reference in theirentirety.

SUBMISSION OF SEQUENCE LISTING ON ASCII TEXT FILE

The content of the following submission on ASCII text file isincorporated herein by reference in its entirety: a computer readableform (CRF) of the Sequence Listing (file name:146392006003SeqListing.txt, date recorded: Dec. 9, 2013, size: 119 KB).

FIELD OF THE INVENTION

The invention relates to anti-CD20 antibodies and their use in thetreatment of B-cell related diseases.

BACKGROUND OF THE INVENTION

Lymphocytes are one of several populations of white blood cells; theyspecifically recognize and respond to foreign antigen. The three majorclasses of lymphocytes are B lymphocytes (B cells), T lymphocytes (Tcells) and natural killer (NK) cells. B lymphocytes are the cellsresponsible for antibody production and provide humoral immunity. Bcells mature within the bone marrow and leave the marrow expressing anantigen-binding antibody on their cell surface. When a naive B cellfirst encounters the antigen for which its membrane-bound antibody isspecific, the cell begins to divide rapidly and its progenydifferentiate into memory B cells and effector cells called “plasmacells”. Memory B cells have a longer life span and continue to expressmembrane-bound antibody with the same specificity as the original parentcell. Plasma cells do not produce membrane-bound antibody but insteadproduce secreted form of the antibody. Secreted antibodies are the majoreffector molecules of humoral immunity.

The CD20 antigen (also called human B-lymphocyte-restricteddifferentiation antigen, Bp35) is a hydrophobic transmembrane proteinwith a molecular weight of approximately 35 kD located on pre-B andmature B lymphocytes (Valentine et al. J. Biol. Chem.264(19):11282-11287 (1989); and Einfeld et al. EMBO J. 7(3):711-717(1988)). The antigen is also expressed on greater than 90% of B cellnon-Hodgkin's lymphomas (NHL) (Anderson et al. Blood 63(6):1424-1433(1984)), but is not found on hematopoietic stem cells, pro-B cells,normal plasma cells or other normal tissues (Tedder et al. J. Immunol.135(2):973-979 (1985)). CD20 is thought to regulate an early step(s) inthe activation process for cell cycle initiation and differentiation(Tedder e al., supra) and possibly functions as a calcium ion channel(Tedder et al. J. Cell. Biochem. 14D:195 (1990)).

Given the expression of CD20 in B cell lymphomas, this antigen has beena useful therapeutic target to treat such lymphomas. There are more than300.000 people in the United States with B-cell NHL and more than 56,000new cases are diagnosed each year. For example, the rituximab (RITUXAN®)antibody which is a genetically engineered chimeric murine/humanmonoclonal antibody directed against human CD20 antigen (commerciallyavailable from Genentech, Inc., South San Francisco. Calif., U.S.) isused for the treatment of patients with relapsed or refractory low-gradeor follicular, CD20 positive. B cell non-Hodgkin's lymphoma. Rituximabis the antibody referred to as “C2B8” in U.S. Pat. No. 5,736,137 issuedApr. 7, 1998 (Anderson et al.) and in U.S. Pat. No. 5,776,456. In vitromechanism of action studies have demonstrated that RITUXAN® binds humancomplement and lyses lymphoid B cell lines through complement-dependentcytotoxicity (CDC) (Reff et al. Blood 83(2):435-445 (1994)).Additionally, it has significant activity in assays forantibody-dependent cellular cytotoxicity (ADCC). In vivo preclinicalstudies have shown that RITUXAN® depletes B cells from the peripheralblood, lymph nodes, and bone marrow of cynomolgus monkeys, presumablythrough complement and cell-mediated processes (Reff et al. Blood83(2):435-445 (1994)). Other anti-CD20 antibodies indicated for thetreatment of NHL include the murine antibody Zevalin™ which is linked tothe radioisotope. Yttrium-90 (IDEC Pharmaceuticals, San Diego, Calif.),Bexxar™ which is a another fully murine antibody conjugated to I-131(Corixa, Wash.).

A major limitation in the use of murine antibodies in human therapy isthe human anti-mouse antibody (HAMA) response (see, e.g., Miller, R. A.et al. “Monoclonal antibody therapeutic trials in seven patients withT-cell lymphoma” Blood, 62:988-995, 1983; and Schroff, R. W., et al.“Human anti-murine immunoglobulin response in patients receivingmonoclonal antibody therapy” Cancer Res., 45:879-885, 1985). Evenchimeric molecules, where the variable (V) domains of rodent antibodiesare fused to human constant (C) regions, are still capable of elicitinga significant immune response (HACA, human anti-chimeric antibody)(Neuberger et al. Nature (Lond.), 314:268-270, 1985). A powerfulapproach to overcome these limitations in the clinical use of monoclonalantibodies is “humanization” of the murine antibody or antibody from anon-human species (Jones et al. Nature (Lond), 321:522-525, 1986;Riechman et al., Nature (Lond), 332:323-327, 1988).

Thus, it is beneficial to produce therapeutic antibodies to the CD20antigen that create minimal or no antigenicity when administered topatients, especially for chronic treatment. The present inventionsatisfies this and other needs. The present invention provides anti-CD20antibodies that overcome the limitations of current therapeuticcompositions as well as offer additional advantages that will beapparent from the detailed description below.

SUMMARY OF THE INVENTION

The present invention provides CD20 binding antibodies or functionalfragments thereof, and their use in the treatment of B-cell associateddiseases. These antibodies are monoclonal antibodies. In specificembodiments, the antibodies that bind CD20 are humanized or chimeric.The humanized 2H7 variants include those that have amino acidsubstitutions in the FR and affinity maturation variants with changes inthe grafted CDRs. The substituted amino acids in the CDR or FR are notlimited to those present in the donor or recipient antibody. In otherembodiments, the anti-CD20 antibodies of the invention further comprisechanges in amino acid residues in the Fc region that lead to improvedeffector function including enhanced CDC and/or ADCC function and B-cellkilling (also referred to herein as B-cell depletion). Other anti-CD20antibodies of the invention include those having specific changes thatimprove stability. In a specific embodiment, the humanized 2H7 variantswith increased stability are as described in example 6 below. Fucosedeficient variants having improved ADCC function in vivo are alsoprovided. In one embodiment, the chimeric anti-CD20 antibody has murineV regions and human C region. One such specific chimeric anti-CD20antibody is Rituxan® (Rituximab®; Genentech, Inc.).

In a preferred embodiment of all of the antibody compositions andmethods of use of this invention, the humanized CD20 binding antibody is2H7.v16 having the light and heavy chain amino acid sequence of SEQ IDNO. 21 and 22, respectively, as shown in FIG. 6 and FIG. 7. Whenreferring to the polypeptide sequences in FIGS. 6, 7 and 8, it should beunderstood that the first 19 or so amino acids that form the secretorysignal sequence are not present in the mature polypeptide. The V regionof all other variants based on version 16 will have the amino acidsequences of v16 except at the positions of amino acid substitutionswhich are indicated in the disclosure. Unless otherwise indicated, the2H7 variants will have the same L chain as that of v16.

The invention provides a humanized antibody that binds human CD20, or anantigen-binding fragment thereof, wherein the antibody is effective todeplete primate B cells in vivo, the antibody comprising in the H chainVariable region (V_(H)) at least a CDR3 sequence of SEQ ID NO. 12 froman anti-human CD20 antibody and substantially the human consensusframework (FR) residues of human heavy chain subgroup III (V_(H)III). Inone embodiment, the primate B cells are from human and Cynomolgusmonkey. In one embodiment, the antibody further comprises the H chainCDR1 sequence of SEQ ID NO, and CDR2 sequence of SEQ ID NO. 11. Inanother embodiment, the preceding antibody comprises the 1, chain CDR1sequence of SEQ ID NO. 4, CDR2 sequence of SEQ ID NO. 5. CDR3 sequenceof SEQ ID NO. 6 with substantially the human consensus framework (FR)residues of human light chain κ subgroup I (VκI). In a preferredembodiment, the FR region In V_(L) has a donor antibody residue atposition 46; in a specific embodiment, FR2 in V_(L) has an amino acidsubstitution of leuL46pro (Leu in the human κI consensus sequencechanged to pro which is present in the corresponding position in m2H7).The VH region further comprises a donor antibody residue at at leastamino acid positions 49, 71 and 73 in the framework. In one embodiment,in the V_(H), the following FR positions in the human heavy chainsubgroup III are substituted: AlaH49Gly in FR2; ArgH71Val and AsnH73Lysin FR3. In other embodiments, the CDR regions in the humanized antibodyfurther comprise amino acid substitutions where the residues arm neitherfrom donor nor recipient antibody.

The antibody of the preceding embodiments can comprise the V_(H)sequence of SEQ ID NO.8 of v16, as shown in FIG. 1B. In a furtherembodiment of the preceding, the antibody further comprises the V_(L)sequence of SEQ ID NO.2 of v16, as shown in FIG. 1A.

In other embodiments, the humanized antibody is 2H7.v31 having the lightand heavy chain amino acid sequence of SEQ ID NO. 21 and 23,respectively, as shown in FIG. 6 and FIG. 8; 2H7.v31 having the heavychain amino acid sequence of SEQ ID NO. 23 as shown in FIG. 8; 2H7.v96with the amino acid substitutions of D56A and N100A in the H chain andS92A in the L chain of v16.

In separate embodiments, the antibody of any of the precedingembodiments further comprises at least one amino acid substitution inthe Fc region that improves ADCC and/or CDC activity over the originalor parent antibody from which it was derived, v.16 being the parentantibody being compared to in most cases, and Rituxan in other cases.One such antibody with improved activity comprises the triple Alaninesubstitution of S298A/E333A/K334A in the Fc region. One antibody havingS298A/E333A/K334A substitution is 2H7.v31 having the heavy chain aminoacid sequence of SEQ ID NO. 23. Antibody 2H7.v114 and 2H7.v115 show atleast 10-fold improved ADCC activity as compared to Rituxan.

In another embodiment, the antibody further comprises at least one aminoacid substitution in the Fc region that decreases CDC activity ascompared to the parent antibody from which it was derived which is v16in most cases. One such antibody with decreased CDC activity as comparedto v16 comprises at least the substitution K322A in the H chain. Thecomparison of ADCC and CDC activity can be assayed as described in theexamples.

In a preferred embodiment, the antibodies of the invention are fulllength antibodies wherein the Vx region is joined to a human IgG heavychain constant region. In preferred embodiments, the IgG is human IgG1or IgG3.

In one embodiment, the CD20 binding antibody is conjugated to acytotoxic agent. In preferred embodiments the cytotoxic agent is a toxinor a radioactive isotope.

In one embodiment, the antibodies of the invention for use intherapeutic or diagnostic purposes are produced in CHO cells.

Also provided is a composition comprising an antibody of any one of thepreceding embodiments, and a carrier. In one embodiment, the carrier isa pharmaceutically acceptable carrier. These compositions can beprovided in an article of manufacture or a kit.

The invention also provided a liquid formulation comprising a humanized2H7 antibody at 20 mg/mL antibody, 10 mM histidine sulfate pH5.8, 60mg/ml sucrose (6%), 0.2 mg/ml polysorbate 20 (0.02%).

The invention also provides an isolated nucleic acid that encodes any ofthe antibodies disclosed herein, including an expression vector forexpressing the antibody.

Another aspect of the invention are host cells comprising the precedingnucleic acids, and host cells that produce the antibody. In a preferredembodiment of the latter, the host cell is a CHO cell. A method ofproducing these antibodies is provided, the method comprising culturingthe host cell that produces the antibody and recovering the antibodyfrom the cell culture.

Yet another aspect of the invention is an article of manufacturecomprising a container and a composition contained therein, wherein thecomposition comprises an antibody of any of the preceding embodiments.For use in treating NHL, the article of manufacture further comprises apackage insert indicating that the composition is used to treatnon-Hodgkin's lymphoma.

A further aspect of the invention is a method of inducing apoptosis in Bcells in vivo, comprising contacting B cells with the antibody of any ofthe preceding, thereby killing the B cells.

The invention also provides methods of treating the diseases disclosedherein by administration of a CD20 binding antibody or functionalfragment thereof, to a mammal such as a human patient suffering from thedisease. In any of the methods for treating an autoimmune disease or aCD20 positive cancer, in one embodiment, the antibody is 2H7.v16 havingthe light and heavy chain amino acid sequence of SEQ ID NO. 21 and 22,respectively, as shown in FIG. 6 and FIG. 7. Thus, one embodiment is amethod of treating a CD20 positive cancer, comprising administering to apatient suffering from the cancer, a therapeutically effective amount ofa humanized CD20 binding antibody of the invention. In preferredembodiments, the CD20 positive cancer is a B cell lymphoma or leukemiaincluding non-Hodgkin's lymphoma (NHL) or lymphocyte predominantHodgkin's disease (LPHD), chronic lymphocytic leukemia (CLL) or SLL. Inone embodiment of the method of treating a B cell lymphoma or leukemia,the antibody is administered at a dosage range of about 275-375 mg/m².In additional embodiments, the treatment method further comprisesadministering to the patient at least one chemotherapeutic agent,wherein for non-Hodgkin's lymphoma (NHL), the chemotherapeutic agent isselected from the group consisting of doxorubicin, cyclophosphamide,vincristine and prednisolone.

Also provided is a method of treating an autoimmune disease, comprisingadministering to a patient suffering from the autoimmune disease, atherapeutically effective amount of the humanized CD20 binding antibodyof any one of the preceding claims. The autoimmune disease is selectedfrom the group consisting of rheumatoid arthritis, juvenile rheumatoidarthritis, systemic lupus erythematosus (SLE), Wegener's disease,inflammatory bowel disease, idiopathic thrombocytopenic purpura (IP),thrombotic thrombocytopenic purpura (TTP), autoimmune thrombocytopenia,multiple sclerosis, psoriasis, IgA nephropathy, IgM polyneuropathies,myasthenia gravis, vasculitis, diabetes mellitus. Reynaud's syndrome,Sjorgen's syndrome and glomerulonephritis. Where the autoimmune diseaseis rheumatoid arthritis, the antibody can be administered in conjunctionwith a second therapeutic agent which is preferably methotrexate.

In these treatment methods, the CD20 binding antibodies can beadministered alone or in conjunction with a second therapeutic agentsuch as a second antibody, or a chemotherapeutic agent or animmunosuppressive agent. The second antibody can be one that binds CD20or a different B cell antigen, or a NK or T cell antigen. In oneembodiment, the second antibody is a radiolabeled anti-CD20 antibody. Inother embodiments, the CD20 binding antibody is conjugated to acytotoxic agent including a toxin or a radioactive isotope.

In another aspect, the invention provides a method of treating anautoimmune disease selected from the group consisting ofDermatomyositis, Wegner's granulomatosis, ANCA, Aplastic anemia,Autoimmune hemolytic anemia (AIHA), factor VIII deficiency, hemophiliaA, Autoimmune neutropenia, Castleman's syndrome, Goodpasture's syndrome,solid organ transplant rejection, graft versus host disease (GVHD), IgMmediated, thrombotic thrombocytopenic purpura (TTP), Hashimoto'sThyroiditis, autoimmune hepatitis, lymphoid interstitial pneumonitis(HIV), bronchiolitis obliterans (non-transplant) vs. NSIP.Guillain-Barre Syndrome, large vessel vasculitis, giant cell(Takayasu's) arteritis, medium vessel vasculitis, Kawasaki's Disease,polyarteritis nodosa, comprising administering to a patient sufferingfrom the disease, a therapeutically effective amount of a CD20 bindingantibody. In one embodiment of this method, the CD20 binding antibody isRituxan®.

The invention also provides an isolated nucleic acid comprising thenucleotide sequence of SEQ ID NO.: 24 of the Cynomolgus monkey CD20(shown in FIG. 19), or a degenerate variant of this sequence. Oneembodiment is an isolated nucleic acid comprising a sequence thatencodes a polypeptide with the amino acid sequence of SEQ ID NO. 25(shown FIG. 20), or SEQ ID NO. 25 (FIG. 20) with conservative amino acidsubstitutions. Another embodiment is a vector comprising the precedingnucleic acid, including an expression vector for expression in a hostcell. Included as well is a host cell comprising the vector. Alsoprovided is an isolated polypeptide comprising the amino acid sequence[SEQ ID NO. 25; FIG. 20] of the Cynomolgus monkey CD20.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A is a sequence alignment comparing the amino acid sequences ofthe light chain variable domain (V_(L)) of each of murine 2H7 (SEQ IDNO. 1), humanized 2H7, v16 variant (SEQ ID NO.2), and human kappa lightchain subgroup I (SEQ ID NO. 3). The CDRs of V_(L) of 2H7 and hu2H7.v16are as follows: CDR1 (SEQ ID NO.4), CDR2 (SEQ ID NO.5), and CDR3 (SEQ IDNO.6).

FIG. 1B is a sequence alignment which compares the V_(H) sequences ofmurine 2H7 (SEQ ID NO. 7), humanized 2H7.v16 variant (SEQ ID NO. 8), andthe human consensus sequence of heavy chain subgroup III (SEQ ID NO. 9).The CDRs of V_(H) of 2H7 and hu2H7.v16 are as follow: CDR1 (SEQ IDNO.10), CDR2 (SEQ ID NO.11), and CDR3 (SEQ ID NO.12).

In FIG. 1A and FIG. 1B, the CDR1, CDR2 and CDR3 in each chain areenclosed within brackets, flanked by the framework regions, FR1-FR4, asindicated. 2H7 refers to the murine 2H7 antibody. The asterisks inbetween two rows of sequences indicate the positions that are differentbetween the two sequences. Residue numbering is according to Kabat etal., Sequences of Immunological Interest. 5th Ed. Public Health Service,National Institutes of Health, Bethesda, Md. (1991), with insertionsshown as a, b, c, d, and e.

FIG. 2A-2E show the sequence of phagemid pVX4 (SEQ ID NO.13) used forconstruction of 2H7 Fab plasmids (see Example 1) as well as the aminoacid sequences of the L chain (SEQ ID NO.14) and H chain (SEQ ID NO.15)of the Fab for the CDR-grafted anti-IFN-t humanized antibody.

FIG. 3A-3E show the sequence of the expression plasmid which encodes thechimeric 2H7.v6.8 Fab (SEQ ID NO.16). The amino acid sequences of the Lchain (SEQ ID NO.17) and H chain (SEQ ID NO.1S) are shown.

FIGS. 4A and 4B show the sequence of the plasmid pDR1 (SEQ ID NO.19:5391 bp) for expression of immunoglobulin light chains as described inExample 1, pDR1 contains sequences encoding an irrelevant antibody, thelight chain of a humanized anti-CD3 antibody (Shalaby et al., J. Exp.Med. 175: 217-225 (1992)), the start and stop codons for which areindicated in bold and underlined.

FIGS. 5A and 5B show the sequence of plasmid pDR2 (SEQ ID NO.20; 6135bp) for expression of immunoglobulin heavy chains as described inExample 1, pDR2 contains sequences encoding an irrelevant antibody, theheavy chain of a humanized anti-CD3 antibody (Shalaby et al., supra),the start and stop codons for which are indicated in bold andunderlined.

FIG. 6 shows the amino acid sequence of the 2H7.v16 complete L chain(SEQ ID NO.21). The first 19 amino acids before DIQ are the secretorysignal sequence not present in the mature polypeptide chain.

FIG. 7 shows the amino acid sequence of the 2H7.v16 complete H chain(SEQ ID NO.22). The first 19 amino acids before EVQ before are thesecretory signal sequence not present in the mature polypeptide chain.Aligning the V_(H) sequence in FIG. 1B (SEQ ID NO. 8) with the completeH chain sequence, the human γ1 constant region is from amino acidposition 114-471 in SEQ ID NO. 22.

FIG. 8 shows the amino acid sequence of the 2H7.v31 complete H chain(SEQ ID NO.23). The first 19 amino acids before EVQ before are thesecretory signal sequence not present in the mature polypeptide chain.The L chain is the same as for 2H7.v16 (see FIG. 6).

FIG. 9 shows the relative stability of 2H7.v16 and 2H7.v73 IgG variantsas described in Example 6. Assay results were normalized to the valuesprior to incubation and reported as percent remaining after incubation.

FIG. 10 is a flow chart summarizing the amino acid changes from themurine 2H7 to a subset of humanized versions up to v75.

FIG. 11 is a summary of mean absolute B-cell count [CD3-/CD40+] in allgroups (2H7 study and Rituxan study combined), as described in Example10.

FIG. 12 shows the results of a representative ADCC assay on fucosedeficient 2H7 variants as described in Example 11.

FIG. 13 shows the results of the Annexin V staining plotted as afunction of antibody concentration. Ramos cells were treated with anirrelevant IgG1 control antibody (Herceptin®; circles), Rituximab(squares), or rhuMAb 2H7.v16 (triangles) in the presence of acrosslinking secondary antibody and were analyzed by FACS. FIGS. 13-15are described in Example 13.

FIG. 14 shows the results of the Annexin V and propidium iodidedouble-staining are plotted as a function of antibody concentration.Ramos cells were treated with an irrelevant IgG1 control antibody(Herceptin®; circles), Rituximab (squares), or rhuMAb 2H7.v16(triangles) in the presence of a crosslinking secondary antibody andwere analyzed by FACS.

FIG. 15 shows the counts (per 10 s) of live, unstained cells are plottedas a function of antibody concentration. Ramos cells were treated withan irrelevant IgG1 control antibody (Herceptin®; circles), Rituximab(squares), or rhuMAb 2H7.v16 (triangles) in the presence of acrosslinking secondary antibody and were analyzed by FACS.

FIGS. 16, 17, 18 show inhibition of Raji cell tumor growth in nude mice,as described in Example 14. Animals were treated weekly (as indicated byvertical arrows; n=g mice per group) for 6 weeks with PBS (control) orwith Rituxan® or rhuMAb 2H7.v16 at 5 mg/kg (FIG. 16), 0.5 mg/kg (FIG.17), or 0.05 mg/kg (FIG. 18).

FIG. 19 shows the nucleotide (SEQ ID NO. 24) and amino acid (SEQ ID NO.25) sequences of Cynomolgus monkey CD20, as described in Example 15.

FIG. 20 shows the amino acid sequence for cynomolgus monkey CD20 (SEQ IDNO. 25). Residues that differ from human CD20 are underlined and thehuman residues (SEQ ID NO. 26) are indicated directly below the monkeyresidue. The putative extracellular domain of the monkey CD20 is in boldtype.

FIG. 21 shows the results of Cynomolgus monkey cells expressing CD20binding to hu2H7.v16, .v31, and Rituxan, as described in Example 15. Theantibodies were assayed for the ability to bind and displaceFTC-conjugated murine 2H7 binding to cynomolgus CD20.

FIG. 22 shows dose escalation schema for rheumatoid arthritis phase I/Iclinical trial.

FIG. 23 shows the vector for expression of 2H7.v16 in CHO cells.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The “CD20” antigen is a non-glycosylated, transmembrane phosphoproteinwith a molecular weight of approximately 35 kD that is found on thesurface of greater than 90% of B cells from peripheral blood or lymphoidorgans. CD20 is expressed during early pre-B cell development andremains until plasma cell differentiation: it is not found on human stemcells, lymphoid progenitor cells or normal plasma cells. CD20 is presenton both normal B cells as well as malignant B cells. Other names forCD20 in the literature include “B-lymphocyte-restricted differentiationantigen” and “Bp35”. The CD20 antigen is described in, for example,Clark and Ledbetter, Adv. Can. Res. 52:81-149 (1989) and Valentine etal. J. Biol. Chem. 264(19):11282-11287 (1989).

The term “antibody” is used in the broadest sense and specificallycovers monoclonal antibodies (including full length monoclonalantibodies), multispecific antibodies (e.g., bispecific antibodies), andantibody fragments so long as they exhibit the desired biologicalactivity or function.

The biological activity of the CD20 binding and humanized CD20 bindingantibodies of the invention will include at least binding of theantibody to human CD20, more preferably binding to human and otherprimate CD20 (including cynomolgus monkey, rhesus monkey, chimpanzees).The antibodies would bind CD20 with a K_(d) value of no higher than1×10⁻⁸, preferably a K_(d) value no higher than about 1×10⁻⁹, and beable to kill or deplete B cells in vivo, preferably by at least 20% whencompared to the appropriate negative control which is not treated withsuch an antibody. B cell depletion can be a result of one or more ofADCC, CDC, apoptosis, or other mechanism. In some embodiments of diseasetreatment herein, specific effector functions or mechanisms may bedesired over others and certain variants of the humanized 2H7 arepreferred to achieve those biological functions, such as ADCC.

“Antibody fragments” comprise a portion of a full length antibody,generally the antigen binding or variable region thereof. Examples ofantibody fragments include Fab, Fab′, F(ab′)₂, and Fv fragments;diabodies; linear antibodies; single-chain antibody molecules; andmultispecific antibodies formed from antibody fragments.

“Fv” is the minimum antibody fragment which contains a completeantigen-recognition and -binding site. This fragment consists of a dimerof one heavy- and one light-chain variable region domain in tight,non-covalent association. From the folding of these two domains emanatesix hypervariable loops (3 loops each from the H and L chain) thatcontribute the amino acid residues for antigen binding and conferantigen binding specificity to the antibody. However, even a singlevariable domain (or half of an Fv comprising only three CDRs specificfor an antigen) has the ability to recognize and bind antigen, althoughat a lower affinity than the entire binding site.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts. Monoclonal antibodies are highly specific, being directedagainst a single antigenic site. Furthermore, in contrast toconventional (polyclonal) antibody preparations which typically includedifferent antibodies directed against different determinants (epitopes),each monoclonal antibody is directed against a single determinant on theantigen. The modifier “monoclonal” indicates the character of theantibody as being obtained from a substantially homogeneous populationof antibodies, and is not to be construed as requiring production of theantibody by any particular method. For example, the monoclonalantibodies to be used in accordance with the present invention may bemade by the hybridoma method first described by Kohler et al., Nature256:495 (1975), or may be made by recombinant DNA methods (see, e.g.,U.S. Pat. No. 4,816,567). The “monoclonal antibodies” may also beisolated from phage antibody libraries using the techniques described inClackson et al., Nature 352:624-628 (1991) and Marks et al. J. Mol.Biol. 222:581-597 (1991), for example.

“Functional fragments” of the CD20 binding antibodies of the inventionare those fragments that retain binding to CD20 with substantially thesame affinity as the intact full length molecule from which they arederived and show biological activity including depleting B cells asmeasured by in vitro or in vivo assays such as those described herein.

The term “variable” refers to the fact that certain segments of thevariable domains differ extensively in sequence among antibodies. The Vdomain mediates antigen binding and defines specificity of a particularantibody for its particular antigen. However, the variability is notevenly distributed across the 110-amino acid span of the variabledomains. Instead, the V regions consist of relatively invariantstretches called framework regions (FRs) of 15-30 amino acids separatedby shorter regions of extreme variability called “hypervariable regions”that are each 9-12 amino acids long. The variable domains of nativeheavy and light chains each comprise four FRs, largely adopting aβ-sheet configuration, connected by three hypervariable regions, whichform loops connecting, and in some cases forming part of, the β-sheetstructure. The hypervariable regions in each chain are held together inclose proximity by the FRs and, with the hypervariable regions from theother chain, contribute to the formation of the antigen-binding site ofantibodies (see Kabat et al., Sequences of Proteins of ImmunologicalInterest, 5th Ed. Public Health Service. National Institutes of Health,Bethesda, Md. (1991)). The constant domains are not involved directly inbinding an antibody to an antigen, but exhibit various effectorfunctions, such as participation of the antibody in antibody dependentcellular cytotoxicity (ADCC).

The term “hypervariable region” when used herein refers to the aminoacid residues of an antibody which are responsible for antigen-binding.The hypervariable region generally comprises amino acid residues from a“complementarity determining region” or “CDR” (e.g. around aboutresidues 24-34 (L1), 50-56 (L2) and 89-97 (L3) in the V_(L), and aroundabout 31-35B (H1), 50-65 (H2) and 95-102 (H3) in the V_(H) (Kabat etal., Sequences of Proteins of Immunological Interest, 5th Ed. PublicHealth Service, National Institutes of Health, Bethesda, Md. (1991))and/or those residues from a “hypervariable loop” (e.g. residues 26-32(L1), 50-52 (L2) and 91-96 (L3) in the V_(L), and 26-32 (H1), 52A-55(H2) and 96-101 (H3) in the V_(H) (Chothia and Lesk J. Mol. Biol.196:901-917 (1987)).

As referred to herein, the “consensus sequence” or consensus V domainsequence is an artificial sequence derived from a comparison of theamino acid sequences of known human immunoglobulin variable regionsequences. Based on these comparisons, recombinant nucleic acidsequences encoding the V domain amino acids that are a consensus of thesequences derived from the human κ and the human H chain subgroup III Vdomains were prepared. The consensus V sequence does not have any knownantibody binding specificity or affinity.

“Chimeric” antibodies (immunoglobulins) have a portion of the heavyand/or light chain identical with or homologous to correspondingsequences in antibodies derived from a particular species or belongingto a particular antibody class or subclass, while the remainder of thechain(s) is identical with or homologous to corresponding sequences inantibodies derived from another species or belonging to another antibodyclass or subclass, as well as fragments of such antibodies, so long asthey exhibit the desired biological activity (U.S. Pat. No. 4,816,567;and Morrison et al., Proc. Natl. Acad Sci. USA 81:6851-6855 (1984)).Humanized antibody as used herein is a subset of chimeric antibodies.

“Humanized” forms of non-human (e.g., murine) antibodies are chimericantibodies which contain minimal sequence derived from non-humanimmunoglobulin. For the most part, humanized antibodies are humanimmunoglobulins (recipient or acceptor antibody) in which hypervariableregion residues of the recipient are replaced by hypervariable regionresidues from a non-human species (donor antibody) such as mouse, rat,rabbit or nonhuman primate having the desired specificity, affinity, andcapacity. In some instances, Fv framework region (FR) residues of thehuman immunoglobulin are replaced by corresponding non-human residues.Furthermore, humanized antibodies may comprise residues which are notfound in the recipient antibody or in the donor antibody. Thesemodifications are made to further refine antibody performance such asbinding affinity. Generally, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the hypervariable loops correspondto those of a non-human immunoglobulin and all or substantially all ofthe FR regions are those of a human immunoglobulin sequence although theFR regions may include one or more amino acid substitutions that improvebinding affinity. The number of these amino acid substitutions in the FRis typically no more than 6 in the H chain, and in the L chain, no morethan 3. The humanized antibody optionally also will comprise at least aportion of an immunoglobulin constant region (Fc), typically that of ahuman immunoglobulin. For further details, see Jones et al., Nature321:522-525 (1986); Reichmann et al., Nature 332:323-329 (1988); andPresta, Curr. Op. Struct. Biol. 2:593-596 (1992).

Antibody “effector functions” refer to those biological activitiesattributable to the Fc region (a native sequence Fe region or amino acidsequence variant Fc region) of an antibody, and vary with the antibodyisotype. Examples of antibody effector functions include: C1q bindingand complement dependent cytotoxicity; Fc receptor binding;antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g. B cell receptor); and B cellactivation.

“Antibody-dependent cell-mediated cytotoxicity” or “ADCC” refers to aform of cytotoxicity in which secreted Ig bound onto Fc receptors (FcRs)present on certain cytotoxic cells (e.g. Natural Killer (NK) cells,neutrophils, and macrophages) enable these cytotoxic effector cells tobind specifically to an antigen-bearing target cell and subsequentlykill the target cell with cytotoxins. The antibodies “arm” the cytotoxiccells and are absolutely required for such killing. The primary cellsfor mediating ADCC, NK cells, express FcγRIII only, whereas monocytesexpress FcγRI, FcγRII and FcγRIII. FcR expression on hematopoietic cellsis summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev.Immunol 9:457-92 (1991). To assess ADCC activity of a molecule ofinterest, an in vitro ADCC assay, such as that described in U.S. Pat.No. 5,500,362 or 5,821,337 may be performed. Useful effector cells forsuch assays Include peripheral blood mononuclear cells (PBMC) andNatural Killer (NK) cells. Alternatively, or additionally, ADC (activityof the molecule of interest may be assessed in vivo, e.g., in a animalmodel such as that disclosed in Clynes et al. PNAS (USA) 95:652-656(1998).

“Fc receptor” or “FcR” describes a receptor that binds to the Fc regionof an antibody. The preferred FcR is a native sequence human FcR.Moreover, a preferred FcR is one which binds an IgG antibody (a gammareceptor) and includes receptors of the FcγRI, FcγRII, and FcγRIIIsubclasses, including allelic variants and alternatively spliced formsof these receptors. FcγRII receptors include FcγRIIA (an “activatingreceptor”) and FcγRIIB (an “inhibiting receptor”), which have similaramino acid sequences that differ primarily in the cytoplasmic domainsthereof. Activating receptor FcγRIIA contains an immunoreceptortyrosine-based activation motif (ITAM) in its cytoplasmic domain.Inhibiting receptor FcγRIIB contains an immunoreceptor tyrosine-basedinhibition motif (ITIM) in its cytoplasmic domain. (see review M, inDaëron. Annu. Rev. Immunol. 15:203-234 (1997)). FcRs are reviewed inRavetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991); Capel et al.,Immunomethods 4:25-34 (1994); and de Haas er al., J. Lab. Clin. Med.126:330-41 (1995). Other FcRs, including those to be identified in thefuture, are encompassed by the term “FcR” herein. The term also includesthe neonatal receptor, FcRn, which is responsible for the transfer ofmaternal IgGs to the fetus (Guyer et al., J. Immunol. 117:587 (1976) andKim er al., J. Immunol. 24:249 (1994)).

WO00/42072 (Presta) describes antibody variants with improved ordiminished binding to FcRs. The content of that patent publication isspecifically incorporated herein by reference. See, also, Shields er al.J. Biol. Chem. 9(2): 6591-6604 (2001).

“Human effector cells” are leukocytes which express one or more FcRs andperform effector functions. Preferably, the cells express at leastFcγRIII and perform ADCC effector function. Examples of human leukocyteswhich mediate ADCC include peripheral blood mononuclear cells (PBMC),natural killer (NK) cells, monocytes, cytotoxic T cells and neutrophils;with PBMCs and NK cells being preferred. The effector cells may beisolated from a native source, e.g. from blood.

“Complement dependent cytotoxicity” or “CDC” refers to the lysis of atarget cell in the presence of complement. Activation of the classicalcomplement pathway is initiated by the binding of the first component ofthe complement system (C1q) to antibodies (of the appropriate subclass)which are bound to their cognate antigen. To assess complementactivation, a CDC assay, e.g. as described in Gazzano-Santoro et al., J.Immunol. Methods 202:163 (1996), may be performed.

Polypeptide variants with altered Fe region amino acid sequences andincreased or decreased C1q binding capability are described in U.S. Pat.No. 6,194,551B1 and WO99/51642. The contents of those patentpublications are specifically incorporated herein by reference. See,also, Idusogie et al. J. Immunol. 164: 4178-4184 (2000).

The N-glycosylation site in IgG is at Asn297 in the CH2 domain. Thepresent invention also provides compositions of a CD20-binding,humanized antibody having a Fc region, wherein about 80-100% (andpreferably about 90-99%) of the antibody in the composition comprises amature core carbohydrate structure which lacks fucose, attached to theFc region of the glycoprotein. Such compositions were demonstratedherein to exhibit a surprising improvement in binding to Fc(RIIIA(F158),which is not as effective as Fc(RIIIA (V158) in interacting with humanIgG. Thus, the compositions herein are anticipated to be superior topreviously described anti-CD20 antibody compositions, especially fortherapy of human patients who express Fc(RIIIA (F158). Fc(RII A (F158)is more common than Fc(RIIIA (V158) in normal, healthy African Americansand Caucasians. See Lehrabecher et al. Blood 94:4220 (1999). The presentapplication further demonstrates the synergistic increase in Fc(RIIIbinding and/or ADCC function that results from combining theglycosylation variations herein with amino acid sequence modification(s)in the Fc region of the glycoprotein.

An “isolated” antibody is one which has been identified and separatedand/or recovered from a component of its natural environment.Contaminant components of its natural environment are materials whichwould interfere with diagnostic or therapeutic uses for the antibody,and may include enzymes, hormones, and other proteinaceous ornonproteinaceous solutes. In preferred embodiments, the antibody will bepurified (1) to greater than 95% E by weight of antibody as determinedby the Lowry method, and most preferably more than 99% by weight, (2) toa degree sufficient to obtain at least 15 residues of N-terminal orinternal amino acid sequence by use of a spinning cup sequenator, or (3)to homogeneity by SDS-PAGE under reducing or nonreducing conditionsusing Coomassie blue or, preferably, silver stain. Isolated antibodyincludes the antibody in situ within recombinant cells since at leastone component of the antibody's natural environment will not be present.Ordinarily, however, isolated antibody will be prepared by at least onepurification step.

An “isolated” nucleic acid molecule is a nucleic acid molecule that isidentified and separated from at least one contaminant nucleic acidmolecule with which it is ordinarily associated in the natural source ofthe antibody nucleic acid. An isolated nucleic acid molecule is otherthan in the form or setting in which it is found in nature. Isolatednucleic acid molecules therefore are distinguished from the nucleic acidmolecule as it exists in natural cells. However, an isolated nucleicacid molecule includes a nucleic acid molecule contained in cells thatordinarily express the antibody where, for example, the nucleic acidmolecule is in a chromosomal location different from that of naturalcells.

The expression “control sequences” refers to DNA sequences necessary forthe expression of an operably linked coding sequence in a particularhost organism. The control sequences that are suitable for prokaryotes,for example, include a promoter, optionally an operator sequence, and aribosome binding site. Eukaryotic cells are known to utilize promoters,polyadenylation signals, and enhancers.

Nucleic acid is “operably linked” when it is placed into a functionalrelationship with another nucleic acid sequence. For example, DNA for apresequence or secretory leader is operably linked to DNA for apolypeptide if it is expressed as a preprotein that participates in thesecretion of the polypeptide; a promoter or enhancer is operably linkedto a coding sequence if it affects the transcription of the sequence; ora ribosome binding site is operably linked to a coding sequence if it ispositioned so as to facilitate translation. Generally, “operably linked”means that the DNA sequences being linked are contiguous, and, in thecase of a secretory leader, contiguous and in reading phase. However,enhancers do not have to be contiguous, linking is accomplished byligation at convenient restriction sites. If such sites do not exist,the synthetic oligonucleotide adaptors or linkers are used in accordancewith conventional practice.

“Vector” includes shuttle and expression vectors. Typically, the plasmidconstruct will also include an origin of replication (e.g., the ColE1origin of replication) and a selectable marker (e.g., ampicillin ortetracycline resistance), for replication and selection, respectively,of the plasmids in bacteria. An “expression vector” refers to a vectorthat contains the necessary control sequences or regulatory elements forexpression of the antibodies including antibody fragment of theinvention, in bacterial or eukaryotic cells. Suitable vectors aredisclosed below.

The cell that produces a humanized CD20 binding antibody of theinvention will include the bacterial and eukaryotic host cells intowhich nucleic acid encoding the antibodies have been introduced.Suitable host cells are disclosed below.

The word “label” when used herein refers to a detectable compound orcomposition which is conjugated directly or indirectly to the antibody.The label may itself be detectable by itself (e.g., radioisotope labelsor fluorescent labels) or, in the case of an enzymatic label, maycatalyze chemical alteration of a substrate compound or compositionwhich is detectable.

An “autoimmune disease” herein is a non-malignant disease or disorderarising from and directed against an individual's own (self) antigensand/or tissues.

As used herein. “B cell depletion” refers to a reduction in B celllevels in an animal or human after drug or antibody treatment, ascompared to the B cell level before treatment. B cell levels aremeasurable using well known assays such as those described in theExperimental Examples. B cell depletion can be complete or partial. Inone embodiment, the depletion of CD20 expressing B cells is at least25%. Not to be limited by any one mechanism, possible mechanisms ofB-cell depletion include ADCC, CDC, apoptosis, modulation of calciumflux or a combination of two or more of the preceding.

The term “cytotoxic agent” as used herein refers to a substance thatinhibits or prevents the function of cells and/or causes destruction ofcells. The term is intended to include radioactive isotopes (e.g., I¹³¹,I¹²⁵, Y⁹⁰ and Re¹⁸⁶), chemotherapeutic agents, and toxins such asenzymatically active toxins of bacterial, fungal, plant or animalorigin, or fragments thereof.

A “chemotherapeutic agent” is a chemical compound useful in thetreatment of cancer. Examples of chemotherapeutic agents includealkalyzing or alkylating agents such as thiotepa and cyclosphosphamide(CYTOXAN™); alkyl sulfonates such as busulfan, improsulfan andpiposulfan; aziridines such as benzodopa, carboquone, meturedopa, anduredopa; ethylenimines and methylamelamines including altretamine,triethylenemelamine, trietylenephosphoramide,triethylenethiophosphaoramide and trimethylolomelamine; nitrogenmustards such as chlorambucil, chlornaphazine, cholophosphamide,estramustine, ifosfamide, mechlorethamine, mechlorethamine oxidehydrochloride, melphalan, novembichin, phenesterine, prednimustine,trofosfamide, uracil mustard; nitrosureas such as carmustine,chlorozotocin, fotemustine, lomustine, nimustine, ranimustine;antibiotics such as aclacinomysins, actinomycin, authramycin, azaserine,bleomycins, cactinomycin, calicheamicin, carabicin, carminomycin,carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin,6-diazo-5-oxo-L-norleucine, doxorubicin (Adriamycin), epiruhicin,esorubicin, idarubicin, marcellomycin, mitomycins, mycophenolic acid,nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin,quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin,ubenimex, zinostatin, rorubicin; anti-metabolites such as methotrexateand 5, fluoouracil (5-FU); folic acid analogues such as denopterin,methotrexate, pteropterin, trimetrexate; purine analogs such astludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidincanalogs such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine,5-FU; androgens such as calusterone, dromostanolone propionate,epitiostanol, mepitiostane, testolactone; anti-adrenals such asaminoglutethimide, mitotane, trilostane; folic acid replenisher such asfrolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinicacid; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine;demecolcine; diaziquone; elfornithine; elliptinium acetate; etoglucid;gallium nitrate; hydroxyurea; lentinan; lonidamine; mitoguazone;mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet; pirarubicin;podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK®; razoxane;sizotlran; spirogermanium; tenuazonic acid; triaziquone; 2,2′,2″-trichlorotriethylamine; urethan; vindesine; dacarbazine:mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine;arabinoside (“Ara-C”); thiotepa; taxoids, e.g. paclitaxel (TAXOL®,Bristol-Myers Squibb Oncology. Princeton. N.J.) and doxetaxel(TAXOTERE®, Rhône-Poulenc Rorer, Antony, France); chlorambucil;gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinumanalogs such as cisplatin and carboplatin; platinum; etoposide (VP-16);ifosfamide; mitomycin C; mitoxantrone; vincristine; vinblastine;vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin;xeloda; ibandronate; CPT-11; topoisomerase inhibitor RFS 2000;difluoromethylornithine (DMFO); retinoic acid; esperamicins;capecitabine; and pharmaceutically acceptable salts, acids orderivatives of any of the above. Also included in this definition areanti-hormonal agents that act to regulate or inhibit hormone action ontumors such as anti-estrogens including for example tamoxifen,raloxifene, aromatase inhibiting 4(5)-imidazoles, 4-hydroxytamoxifen,trioxifene, keoxifene, LY117018, onapristone, and toremifene (Fareston);anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide,and goserelin; other chemotherapeutic agents such as prednisolone.Pharmaceutically acceptable salts, acids or derivatives of any of theabove are included.

“Treating” or “treatment” or “alleviation” refers to both therapeutictreatment and prophylactic or preventative measures, wherein the objectis to prevent or slow down (lessen) the targeted pathologic condition ordisorder. A subject is successfully “treated” for a CD20 positive canceror an autoimmune disease if, after receiving a therapeutic amount of aCD20 binding antibody of the invention according to the methods of thepresent invention, the subject shows observable and/or measurablereduction in or absence of one or more signs and symptoms of theparticular disease. For example, for cancer, reduction in the number ofcancer cells or absence of the cancer cells; reduction in the tumorsize; inhibition (i.e., slow to some extent and preferably stop) oftumor metastasis; inhibition, to some extent, of tumor growth; increasein length of remission, and/or relief to some extent, one or more of thesymptoms associated with the specific cancer, reduced morbidity andmortality, and improvement in quality of life issues. Reduction of thesigns or symptoms of a disease may also be felt by the patient.Treatment can achieve a complete response, defined as disappearance ofall signs of cancer, or a partial response, wherein the size of thetumor is decreased, preferably by more than 50 percent, more preferablyby 75%. A patient is also considered treated if the patient experiencesstable disease. In a preferred embodiment, the cancer patients are stillprogression-free in the cancer after one year, preferably after 15months. These parameters for assessing successful treatment andimprovement in the disease are readily measurable by routine proceduresfamiliar to a physician of appropriate skill in the art.

A “therapeutically effective amount” refers to an amount of an antibodyor a drug effective to “treat” a disease or disorder in a subject. Inthe case of cancer, the therapeutically effective amount of the drug mayreduce the number of cancer cells; reduce the tumor size; inhibit (i.e.,slow to some extent and preferably stop) cancer cell infiltration intoperipheral organs; inhibit (i.e., slow to some extent and preferablystop) tumor metastasis; inhibit, to some extent, tumor growth; and/orrelieve to some extent one or more of the symptoms associated with thecancer. See preceding definition of “treating”.

“Chronic” administration refers to administration of the agent(s) in acontinuous mode as opposed to an acute mode, so as to maintain theinitial therapeutic effect (activity) for an extended period of time.“Intermittent” administration is treatment that is not consecutivelydone without interruption, but rather is cyclic in nature.

Compositions and Methods of the Invention

The invention provides humanized antibodies that bind human CD20, andpreferably other primate CD20 as well, comprising a H chain having atleast one, preferably two or all of the H chain CDRs of a non-humanspecies anti-human CD20 antibody (donor antibody), and substantially allof the framework residues of a human consensus antibody as the recipientantibody. The donor antibody can be from various non-human speciesincluding mouse, rat, guinea pig, goat, rabbit, horse, primate but mostfrequently will be a murine antibody. “Substantially all” in thiscontext is meant that the recipient FR regions in the humanized antibodymay include one or more amino acid substitutions not originally presentin the human consensus FR sequence. These FR changes may compriseresidues not found in the recipient or the donor antibody.

In one embodiment, the donor antibody is the murine 2H7 antibody, the Vregion including the CDR and FR sequences of each of the H and L chainsof which are shown in FIGS. 1A and 1B. In a specific embodiment, theresidues for the human Fab framework correspond to the consensussequence of human Vκ subgroup I and of V_(H) subgroup III, theseconsensus sequences are shown in FIG. 1A and FIG. 1B, respectively. Thehumanized 2H7 antibody of the invention will have at least one of theCDRs in the H chain of the murine donor antibody. In one embodiment, thehumanized 2H7 antibody that hinds human CD20 comprises the CDRs of boththe H and L chains of the donor antibody.

In a full length antibody, the humanized CD20 binding antibody of theinvention will comprise a humanized V domain joined to a C domain of ahuman immunoglobulin. In a preferred embodiment, the H chain C region isfrom human IgG, preferably IgG1 or IgG3. The L chain C domain ispreferably from human κ chain.

Unless indicated otherwise, a humanized 2H7 antibody version herein willhave the V and C domain sequences of 2H7.v16 L chain (FIG. 6, SEQ ID NO.21) and H chain (FIG. 7, SEQ ID NO. 22) except at the positions of aminoacid substitutions or changes indicated in the experimental examplesbelow.

The humanized CD20 binding antibodies will bind at least human CD20 andpreferably bind other primate CD20 such as that of monkeys includingcynomolgus and rhesus monkeys, and chimpanzees. The sequence of thecynomolgus monkey CD20 is disclosed in Example 15 and FIG. 19

The biological activity of the CD20 binding antibodies and humanizedCD20 binding antibodies of the invention will include at least bindingof the antibody to human CD20, more preferably binding to human andprimate CD20 (including cynomolgus monkey, rhesus monkey, chimpanzees),with a K_(d) value of no higher than 1×10⁻⁸, preferably a K_(d) value nohigher than about 1×10⁻⁹, even more preferably a K_(d) value no higherthan about 1×10⁻¹⁰, and be able to kill or deplete B cells in vitro orin vivo, preferably by at least 20% when compared to the baseline levelor appropriate negative control which is not treated with such anantibody.

The desired level of B cell depletion will depend on the disease. Forthe treatment of a CD20 positive cancer, it may be desirable to maximizethe depletion of the B cells which are the target of the anti-CD20antibodies of the invention. Thus, for the treatment of a CD20 positiveB cell neoplasm, it is desirable that the B cell depletion be sufficientto at least prevent progression of the disease which can be assessed bythe physician of skill in the art, e.g., by monitoring tumor growth(size), proliferation of the cancerous cell type, metastasis, othersigns and symptoms of the particular cancer. Preferably, the B celldepletion is sufficient to prevent progression of disease for at least 2months, more preferably 3 months, even more preferably 4 months, morepreferably 5 months, even more preferably 6 or more months. In even morepreferred embodiments, the B cell depletion is sufficient to increasethe time in remission by at least 6 months, more preferably 9 months,more preferably one year, more preferably 2 years, more preferably 3years, even more preferably 5 or more years. In a most preferredembodiment, the B cell depletion is sufficient to cure the disease. Inpreferred embodiments, the B cell depletion in a cancer patient is atleast about 75% and more preferably, 80%, 85%, 90%, 95%, 99% and even100% of the baseline level before treatment.

For treatment of an autoimmune disease, it may be desirable to modulatethe extent of B cell depletion depending on the disease and/or theseverity of the condition in the individual patient, by adjusting thedosage of CD20 binding antibody. Thus. B cell depletion can but does nothave to be complete. Or, total B cell depletion may be desired ininitial treatment but in subsequent treatments, the dosage may beadjusted to achieve only partial depletion. In one embodiment, the Bcell depletion is at least 20%, i.e., 80% or less of CD20 positive Bcells remain as compared to the baseline level before treatment. Inother embodiments, B cell depletion is 25%, 30%, 40%, 50%, 60%, 70% orgreater. Preferably, the B cell depletion is sufficient to haltprogression of the disease, more preferably to alleviate the signs andsymptoms of the particular disease under treatment, even more preferablyto cure the disease.

The invention also provides bispecific CD20 binding antibodies whereinone arm of the antibody has a humanized H and L chain of the humanizedCD20 binding antibody of the invention, and the other arm has V regionbinding specificity for a second antigen. In specific embodiments, thesecond antigen is selected from the group consisting of CD3, CD64,CD32A. CD16, NKG2D or other NK activating ligands.

In comparison with Rituxan (rituximab), v16 exhibits about 2 to 5 foldincreased ADCC potency, ˜3-4 fold decreased CDC than Rituxan.

Antibody Production

Monoclonal Antibodies

Monoclonal antibodies may be made using the hybridoma method firstdescribed by Kohler et al, Nature, 256:495 (1975), or may be made byrecombinant DNA methods (U.S. Pat. No. 4,816,567).

In the hybridoma method, a mouse or other appropriate host animal, suchas a hamster, is immunized as described above to elicit lymphocytes thatproduce or are capable of producing antibodies that will specificallybind to the protein used for immunization. Alternatively, lymphocytesmay be immunized in vitro. After immunization, lymphocytes are isolatedand then fused with a myeloma cell line using a suitable fusing agent,such as polyethylene glycol, to form a hybridoma cell (Goding,Monoclonal Antibodies: Principles and Practice, pp. 59-103 (AcademicPress, 1986)).

The hybridoma cells thus prepared are seeded and grown in a suitableculture medium which medium preferably contains one or more substancesthat inhibit the growth or survival of the unfused, parental myelomacells (also referred to as fusion partner). For example, if the parentalmyeloma cells lack the enzyme hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT), the selective culture medium for thehybridomas typically will include hypoxanthine, aminopterin, andthymidine (HAT medium), which substances prevent the growth ofHGPRT-deficient cells.

Preferred fusion partner myeloma cells are those that fuse efficiently,support stable high-level production of antibody by the selectedantibody-producing cells, and are sensitive to a selective medium thatselects against the unfused parental cells. Preferred myeloma cell linesare murine myeloma lines, such as those derived from MOPC-21 and MPC-11mouse tumors available from the Salk Institute Cell Distribution Center,San Diego, Calif. USA, and SP-2 and derivatives e.g., X63-Ag8-653 cellsavailable from the American Type Culture Collection, Rockville, Md. USA.Human myeloma and mouse-human heteromyeloma cell lines also have beendescribed for the production of human monoclonal antibodies (Korbor, J.Immunol., 133:3001 (1984); and Brodeur et al., Monoclonal AntibodyProduction Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc.,New York, 1987)).

Culture medium in which hybridoma cells are growing is assayed forproduction of monoclonal antibodies directed against the antigen.Preferably, the binding specificity of monoclonal antibodies produced byhybridoma cells is determined by immunoprecipitation or by an in vitrobinding assay, such as radioimmunoassay (RIA) or enzyme-linkedimmunosorbent assay (ESA).

The binding affinity of the monoclonal antibody can, for example, bedetermined by the Scatchard analysis described in Munson et al., Anal.Biochem., 107:220 (1980).

Once hybridoma cells that produce antibodies of the desired specificity,affinity, and/or activity are identified, the clones may be subcloned bylimiting dilution procedures and grown by standard methods (Goding,Monoclonal Antibodies: Principles and Practice, pp. 59-103 (AcademicPress, 1986)). Suitable culture media for this purpose include, forexample, D-MEM or RPMI-1640 medium. In addition, the hybridoma cells maybe grown in vivo as ascites tumors in an animal e.g, by i.p. injectionof the cells into mice.

The monoclonal antibodies secreted by the subclones are suitablyseparated from the culture medium, ascites fluid, or serum byconventional antibody purification procedures such as, for example,affinity chromatography (e.g., using protein A or protein G-Sepharose)or ion-exchange chromatography, hydroxylapatite chromatography, gelelectrophoresis, dialysis, etc.

DNA encoding the monoclonal antibodies is readily isolated and sequencedusing conventional procedures (e.g., by using oligonucleotide probesthat are capable of binding specifically to genes encoding the heavy andlight chains of murine antibodies). The hybridoma cells serve as apreferred source of such DNA. Once isolated, the DNA may be placed intoexpression vectors, which are then transfected into host cells such asE. coli cells, simian COS cells, Chinese Hamster Ovary (CHO) cells, ormyeloma cells that do not otherwise produce antibody protein, to obtainthe synthesis of monoclonal antibodies in the recombinant host cells.Review articles on recombinant expression in bacteria of DNA encodingthe antibody include Skerra et al., Curr. Opinion in Immunol., 5:256-262(1993) and Plückthun, Immunol. Revs., 130:151-188 (1992).

In a further embodiment, monoclonal antibodies or antibody fragments canbe isolated from antibody phage libraries generated using the techniquesdescribed in McCafferty et al., Nature, 348:552.554 (1990). Clackson etal. Nature, 352:624-628 (1991) and Marks et al. J. Mol. Biol.,222:581-597 (1991) describe the isolation of murine and humanantibodies, respectively, using phage libraries. Subsequent publicationsdescribe the production of high affinity (nM range) human antibodies bychain shuffling (Marks et al., Bio/Technology, 10:779-783 (1992)), aswell as combinatorial infection and in vivo recombination as a strategyfor constructing very large phage libraries (Waterhouse et al. Nuc.Acids. Res., 21:2265-2266 (1993)). Thus, these techniques are viablealternatives to traditional monoclonal antibody hybridoma techniques forisolation of monoclonal antibodies.

The DNA that encodes the antibody may be modified to produce chimeric orfusion antibody polypeptides, for example, by substituting human heavychain and light chain constant domain (C_(H) and C_(L)) sequences forthe homologous murine sequences (U.S. Pat. No. 4,816,567; and Morrison,et al., Proc. Natl Acad Sci. USA, 81:6851 (1984)), or by fusing theimmunoglobulin coding sequence with all or part of the coding sequencefor a non-immunoglobulin polypeptide (heterologous polypeptide). Thenon-immunoglobulin polypeptide sequences can substitute for the constantdomains of an antibody, or they are substituted for the variable domainsof one antigen-combining site of an antibody to create a chimericbivalent antibody comprising one antigen-combining site havingspecificity for an antigen and another antigen-combining site havingspecificity for a different antigen.

Humanized Antibodies

Methods for humanizing non-human antibodies have been described in theart. Preferably, a humanized antibody has one or more amino acidresidues introduced into it from a source which is non-human. Thesenon-human amino acid residues are often referred to as “import”residues, which are typically taken from an “import” variable domain.Humanization can be essentially performed following the method of Winterand co-workers (Jones et al., Nature, 321:522-525 (1986). Reichmann etal., Nature, 332:323-327 (1988); Verhoeyen et al., Science,239:1534.1536 (1988)), by substituting hypervariable region sequencesfor the corresponding sequences of a human antibody. Accordingly, such“humanized” antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567)wherein substantially less than an intact human variable domain has beensubstituted by the corresponding sequence from a non-human species. Inpractice, humanized antibodies are typically human antibodies in whichsome hypervariable region residues and possibly some FR residues aresubstituted by residues from analogous sites in rodent antibodies.

The choice of human variable domains, both light and heavy, to be usedin making the humanized antibodies Is very important to reduceantigenicity and HAMA response (human anti-mouse antibody) when theantibody is intended for human therapeutic use. According to theso-called “best-fit” method, the sequence of the variable domain of arodent antibody is screened against the entire library of known humanvariable domain sequences. The human V domain sequence which is closestto that of the rodent is identified and the human framework region (FR)within it accepted for the humanized antibody (Sims et al., J. Immunol.,151:2296 (1993); Chothia et al., J. Mol. Biol., 196:901 (1987)). Anothermethod uses a particular framework region derived from the consensussequence of all human antibodies of a particular subgroup of light orheavy chains. The same framework may be used for several differenthumanized antibodies (Carter et al., Proc. Natl. Acad Sci. USA, 89:4285(1992); Presta et al., J. Immunol., 151:2623 (1993)).

It is further important that antibodies be humanized with retention ofhigh binding affinity for the antigen and other favorable biologicalproperties. To achieve this goal, according to a preferred method,humanized antibodies are prepared by a process of analysis of theparental sequences and various conceptual humanized products usingthree-dimensional models of the parental and humanized sequences.Three-dimensional immunoglobulin models are commonly available and arefamiliar to those skilled in the art. Computer programs are availablewhich illustrate and display probable three-dimensional conformationalstructures of selected candidate immunoglobulin sequences. Inspection ofthese displays permits analysis of the likely role of the residues inthe functioning of the candidate immunoglobulin sequence, i.e., theanalysis of residues that influence the ability of the candidateimmunoglobulin to bind its antigen. In this way, FR residues can beselected and combined from the recipient and import sequences so thatthe desired antibody characteristic, such as increased affinity for thetarget antigen(s), is achieved. In general, the hypervariable regionresidues are directly and most substantially involved in influencingantigen binding.

The humanized antibody may be an antibody fragment, such as a Fab, whichis optionally conjugated with one or more cytotoxic agent(s) in order togenerate an immunoconjugate. Alternatively, the humanized antibody maybe an full length antibody, such as an full length IgG1 antibody.

Human Antibodies and Phage Display Methodology

As an alternative to humanization, human antibodies can be generated.For example, it is now possible to produce transgenic animals (e.g.,mice) that are capable, upon immunization, of producing a fullrepertoire of human antibodies in the absence of endogenousimmunoglobulin production. For example, it has been described that thehomozygous deletion of the antibody heavy-chain joining region (J_(H))gene in chimeric and germ-line mutant mice results in completeinhibition of endogenous antibody production. Transfer of the humangerm-line immunoglobulin gene array into such germ-line mutant mice willresult in the production of human antibodies upon antigen challenge.See, e.g., Jakobovits et al., Proc. Natl. Acad Sci. USA, 90:2551 (1993);Jakobovits et al., Nature, 362:255-258 (1993); Bruggemann et al., Yearin Immuno., 7:33 (1993); U.S. Pat. Nos. 5,545,806, 5,569,825, 5,591.669(all of GenPharm); U.S. Pat. No. 5,545,807; and WO 97/17852.

Alternatively, phage display technology (McCafferty et al., Nature348:552-553 [1990]) can be used to produce human antibodies and antibodyfragments in vitro, from immunoglobulin variable (V) domain generepertoires from unimmunized donors. According to this technique,antibody V domain genes are cloned in-frame into either a major or minorcoat protein gene of a filamentous bacteriophage, such as M13 or fd, anddisplayed as functional antibody fragments on the surface of the phageparticle. Because the filamentous particle contains a single-strandedDNA copy of the phage genome, selections based on the functionalproperties of the antibody also result in selection of the gene encodingthe antibody exhibiting those properties. Thus, the phage mimics some ofthe properties of the B-cell. Phage display can be performed in avariety of formats, reviewed in, e.g., Johnson, Kevin S. and Chiswell,David J., Current Opinion in Structural Biology 3:564-571 (1993).Several sources of V-gene segments can be used for phage display.Clackson et al., Nature, 352:624-628 (1991) isolated a diverse array ofanti-oxazolone antibodies from a small random combinatorial library of Vgenes derived from the spleens of immunized mice. A repertoire of Vgenes from unimmunized human donors can be constructed and antibodies toa diverse array of antigens (including self-antigens) can be isolatedessentially following the techniques described by Marks e al., J. Mol.Biol. 222:581-597 (1991), or Griffith et al., EMBO J. 12:725-734 (1993).See, also, U.S. Pat. Nos. 5,565,332 and 5,573,905.

As discussed above, human antibodies may also be generated by in vitroactivated B cells (see U.S. Pat. Nos. 5,567,610 and 5,229,275).

Antibody Fragments

In certain circumstances there are advantages of using antibodyfragments, rather than whole antibodies. The smaller size of thefragments allows for rapid clearance, and may lead to improved access tosolid tumors.

Various techniques have been developed for the production of antibodyfragments. Traditionally, these fragments were derived via proteolyticdigestion of intact antibodies (see, e.g., Morimoto et al., Journal ofBiochemical and Biophysical Methods 24:107-117 (1992); and Brennan etal., Science, 229:81 (1985)). However, these fragments can now beproduced directly by recombinant host cells. Fab, Fv and ScFv antibodyfragments can all be expressed in and secreted from E. coli, thusallowing the facile production of large amounts of these fragments.Antibody fragments can be isolated from the antibody phage librariesdiscussed above. Alternatively, Fab′-SH fragments can be directlyrecovered from E. coli and chemically coupled to form F(ab′)₂ fragments(Carter et al., Bio/Technology 10:163-167 (1992)). According to anotherapproach, F(ab′)₂ fragments can be isolated directly from recombinanthost cell culture. Fab and F(ab′)₂ fragment with increased in vivohalf-life comprising a salvage receptor binding epitope residues aredescribed in U.S. Pat. No. 5,869,046. Other techniques for theproduction of antibody fragments will be apparent to the skilledpractitioner. In other embodiments, the antibody of choice is a singlechain Fv fragment (scFv). See WO 93/16185; U.S. Pat. Nos. 5,571,894; and5,587,458. Fv and sFv are the only species with intact combining sitesthat are devoid of constant regions; thus, they are suitable for reducednonspecific binding during in vivo use, sFv fusion proteins may beconstructed to yield fusion of an effector protein at either the aminoor the carboxy terminus of an sFv. See Antibody Engineering, ed.Borrebaeck, supra. The antibody fragment may also be a “linearantibody”. e.g., as described in U.S. Pat. No. 5,641,870 for example.Such linear antibody fragments may be monospecific or bispecific.

Bispecific Antibodies

Bispecific antibodies are antibodies that have binding specificities forat least two different epitopes. Exemplary bispecific antibodies maybind to two different epitopes of the CD20 protein. Other suchantibodies may combine a CD20 binding site with a binding site foranother protein. Alternatively, an anti-CD20 arm may be combined with anarm which binds to a triggering molecule on a leukocyte such as a T-cellreceptor molecule (e.g. CD3), or Fc receptors for IgG Fc-R), such asFcγRI (CD64), FcγRII (CD32) and FcγRIII (CD16), or NK2D or other NK cellactivating ligand, so as to focus and localize cellular defensemechanisms to the CD20-expressing cell. Bispecific antibodies may alsobe used to localize cytotoxic agents to cells which express CD20. Theseantibodies possess a CD20-binding arm and an arm which binds thecytotoxic agent (e.g. saporin, anti-interferon-α, vinca alkaloid, ricinA chain, methotrexate or radioactive isotope hapten). Bispecificantibodies can be prepared as full length antibodies or antibodyfragments (e.g. F(ab′)₂ bispecific antibodies).

WO 96/16673 describes a bispecific anti-ErbB2/anti-FcγRIII antibody andU.S. Pat. No. 5,837,234 discloses a bispecific anti-ErbB2/anti-FcγRIantibody. A bispecific anti-ErbB2/Fcα antibody is shown in WO98/02463.U.S. Pat. No. 5,821,337 teaches a bispecific anti-ErbB2/anti-CD3antibody.

Methods for making bispecific antibodies are known in the art.Traditional production of full length bispecific antibodies is based onthe co-expression of two immunoglobulin heavy chain-light chain pairs,where the two chains have different specificities (Millstein et al.,Nature. 305:537-539 (1983)). Because of the random assortment ofimmunoglobulin heavy and light chains, these hybridomas (quadromas)produce a potential mixture of 10 different antibody molecules, of whichonly one has the correct bispecific structure. Purification of thecorrect molecule, which is usually done by affinity chromatographysteps, is rather cumbersome, and the product yields are low. Similarprocedures are disclosed in WO 93/08829, and in Traunecker et al., EMBOJ., 10:3655-3659 (1991).

According to a different approach, antibody variable domains with thedesired binding specificities (antibody-antigen combining sites) arefused to immunoglobulin constant domain sequences. Preferably, thefusion is with an Ig heavy chain constant domain, comprising at leastpart of the hinge, C_(H)2, and C_(H)3 regions. It is preferred to havethe first heavy-chain constant region (C_(H)1) containing the sitenecessary for light chain bonding, present in at least one of thefusions. DNAs encoding the immunoglobulin heavy chain fusions and, ifdesired, the immunoglobulin light chain, are inserted into separateexpression vectors, and are co-transfected into a suitable host cell.This provides for greater flexibility in adjusting the mutualproportions of the three polypeptide fragments in embodiments whenunequal ration of the three polypeptide chains used in the constructionprovide the optimum yield of the desired bispecific antibody. It is,however, possible to insert the coding sequences for two or all threepolypeptide chains into a single expression vector when the expressionof at least two polypeptide chains in equal ratios results in highyields or when the ratios have no significant affect on the yield of thedesired chain combination.

In a preferred embodiment of this approach, the bispecific antibodiesare composed of a hybrid immunoglobulin heavy chain with a first bindingspecificity in one arm, and a hybrid immunoglobulin heavy chain-lightchain pair (providing a second binding specificity) in the other arm. Itwas found that this asymmetric structure facilitates the separation ofthe desired bispecific compound from unwanted immunoglobulin chaincombinations, as the presence of an immunoglobulin light chain in onlyone half of the bispecific molecule provides for a facile way ofseparation. This approach is disclosed in WO 94/04690. For furtherdetails of generating bispecific antibodies see, for example, Suresh etal., Methods in Enzymology, 121:210 (1986).

According to another approach described in U.S. Pat. No. 5,731,168, theinterface between a pair of antibody molecules can be engineered tomaximize the percentage of heterodimers which are recovered fromrecombinant cell culture. The preferred interface comprises at least apart of the C_(H)3 domain. In this method, one or more small amino acidside chains from the interface of the first antibody molecule arereplaced with larger side chains (e.g. tyrosine or tryptophan).Compensatory “cavities” of identical or similar size to the large sidechain(s) are created on the interface of the second antibody molecule byreplacing large amino acid side chains with smaller ones (e.g. alanineor threonine). This provides a mechanism for increasing the yield of theheterodimer over other unwanted end-products such as homodimers.

Bispecific antibodies include cross-linked or “heteroconjugate”antibodies. For example, one of the antibodies in the heteroconjugatecan be coupled to avidin, the other to biotin. Such antibodies have, forexample, been proposed to target immune system cells to unwanted cells(U.S. Pat. No. 4,676,980), and for treatment of HIV infection (WO91/00360, WO 92/200373, and EP 03089). Heteroconjugate antibodies may bemade using any convenient cross-linking methods. Suitable cross-linkingagents are well known in the art, and are disclosed in U.S. Pat. No.4,676,980, along with a number of cross-linking techniques.

Techniques for generating bispecific antibodies from antibody fragmentshave also been described in the literature. For example, bispecificantibodies can be prepared using chemical linkage. Brennan et al.,Science, 229: 81(1985) describe a procedure wherein intact antibodiesare proteolytically cleaved to generate F(ab′)₂ fragments. Thesefragments are reduced in the presence of the dithiol complexing agent,sodium arsenite, to stabilize vicinal dithiols and preventintermolecular disulfide formation. The Fab′ fragments generated arethen converted to thionitrobenzoate (TNB) derivatives. One of theFab′-TNB derivatives is then reconverted to the Fab′-thiol by reductionwith mercaptoethylamine and is mixed with an equimolar amount of theother Fab′-TNB derivative to form the bispecific antibody. Thebispecific antibodies produced can be used as agents for the selectiveimmobilization of enzymes.

Recent progress has facilitated the direct recovery of Fab′-Si fragmentsfrom E. coli, which can be chemically coupled to form bispecificantibodies. Shalaby e al., J. Exp. Med. 175: 217-225 (1992) describe theproduction of a fully humanized bispecific antibody F(ab′)₂ molecule.Each Fab′ fragment was separately secreted from E. coli and subjected todirected chemical coupling in vitro to form the bispecific antibody. Thebispecific antibody thus formed was able to bind to cells overexpressingthe ErbB2 receptor and normal human T cells, as well as trigger thelytic activity of human cytotoxic lymphocytes against human breast tumortargets.

Various techniques for making and isolating bispecific antibodyfragments directly from recombinant cell culture have also beendescribed. For example, bispecific antibodies have been produced usingleucine zippers. Kostelny er al., J. Immunol., 148(5):1547-1553 (1992).The leucine zipper peptides from the Fos and Jun proteins were linked tothe Fab′ portions of two different antibodies by gene fusion. Theantibody homodimers were reduced at the hinge region to form monomersand then re-oxidized to form the antibody heterodimers. This method canalso be utilized for the production of antibody homodimers. The“diabody” technology described by Hollinger et al., Proc. Natl. Acad.Sci. USA, 90:6444-6448 (1993) has provided an alternative mechanism formaking bispecific antibody fragments. The fragments comprise a V_(H)connected to a V_(L) by a linker which is too short to allow pairingbetween the two domains on the same chain. Accordingly, the V_(H) andV_(L) domains of one fragment are forced to pair with the complementaryV_(L) and V_(H) domains of another fragment, thereby forming twoantigen-binding sites. Another strategy for making bispecific antibodyfragments by the use of single-chain Fv (sFv) dimers has also beenreported. See Gruber et al., J. Immunol., 152:5368 (1994).

Antibodies with more than two valencies are contemplated. For example,trispecific antibodies can be prepared. Tutt e al. J. Immunol. 147: 60(1991).

Multivalent Antibodies

A multivalent antibody may be internalized (and/or catabolized) fasterthan a bivalent antibody by a cell expressing an antigen to which theantibodies bind. The antibodies of the present invention can bemultivalent antibodies (which are other than of the IgM class) withthree or more antigen binding sites (e.g. tetravalent antibodies), whichcan be readily produced by recombinant expression of nucleic acidencoding the polypeptide chains of the antibody. The multivalentantibody can comprise a dimerization domain and three or more antigenbinding sites. The preferred dimerization domain comprises (or consistsof) an Fc region or a hinge region. In this scenario, the antibody willcomprise an Fc region and three or more antigen binding sitesamino-terminal to the Fc region. The preferred multivalent antibodyherein comprises (or consists of) three to about eight, but preferablyfour, antigen binding sites. The multivalent antibody comprises at leastone polypeptide chain (and preferably two polypeptide chains), whereinthe polypeptide chain(s) comprise two or more variable domains. Forinstance, the polypeptide chain(s) may compriseVD1-(X1)_(n)-VD2-(X2)_(n)-Fc, wherein VD1 is a first variable domain,VD2 is a second variable domain, Fc is one polypeptide chain of an Feregion, X1 and X2 represent an amino acid or polypeptide, and n is 0or 1. For instance, the polypeptide chain(s) may comprise:VH-CH1-flexible linker-VH-CH1-Fc region chain; or VH-CH1-VH-CH1-Fcregion chain. The multivalent antibody herein preferably furthercomprises at least two (and preferably four) light chain variable domainpolypeptides. The multivalent antibody herein may, for instance,comprise from about two to about eight light chain variable domainpolypeptides. The light chain variable domain polypeptides contemplatedhere comprise a light chain variable domain and, optionally, furthercomprise a CL domain.

Other Amino Acid Sequence Modifications

Amino acid sequence modification(s) of the CD2 binding antibodiesdescribed herein are contemplated. For example, it may be desirable toimprove the binding affinity and/or other biological properties of theantibody. Amino acid sequence variants of the anti-CD20 antibody areprepared by introducing appropriate nucleotide changes into theanti-CD20 antibody nucleic acid, or by peptide synthesis. Suchmodifications include, for example, deletions from, and/or insertionsinto and/or substitutions of, residues within the amino acid sequencesof the anti-CD20 antibody. Any combination of deletion, insertion, andsubstitution is made to arrive at the final construct, provided that thefinal construct possesses the desired characteristics. The amino acidchanges also may alter post-translational processes of the anti-CD20antibody, such as changing the number or position of glycosylationsites.

A useful method for identification of certain residues or regions of theanti-CD20 antibody that are preferred locations for mutagenesis iscalled “alanine scanning mutagenesis” as described by Cunningham andWells in Science, 244:1081-1085 (1989). Here, a residue or group oftarget residues are identified (e.g., charged residues such as arg, asp,his, lys, and glu) and replaced by a neutral or negatively charged aminoacid (most preferably alanine or polyalanine) to affect the interactionof the amino acids with CD20 antigen. Those amino acid locationsdemonstrating functional sensitivity to the substitutions then arerefined by introducing further or other variants at, or for, the sitesof substitution. Thus, while the site for introducing an amino acidsequence variation is predetermined, the nature of the mutation per seneed not be predetermined. For example, to analyze the performance of amutation at a given site, ala scanning or random mutagenesis isconducted at the target codon or region and the expressed anti-CD20antibody variants are screened for the desired activity.

Amino acid sequence insertions include amino- and/or carboxyl-terminalfusions ranging in length from one residue to polypeptides containing ahundred or more residues, as well as intrasequence insertions of singleor multiple amino acid residues. Examples of terminal insertions includean anti-CD20 antibody with an N-terminal methionyl residue or theantibody fused to a cytotoxic polypeptide. Other insertional variants ofthe anti-CD20 antibody molecule include the fusion to the N- orC-terminus of the anti-CD20 antibody to an enzyme (e.g. for ADEPT) or apolypeptide which increases the serum half-life of the antibody.

Another type of variant is an amino acid substitution variant. Thesevariants have at least one amino acid residue in the anti-CD2 antibodymolecule replaced by a different residue. The sites of greatest interestfor substitutional mutagenesis include the hypervariable regions, but FRalterations are also contemplated. Conservative substitutions are shownin the Table below under the heading of “preferred substitutions”. Ifsuch substitutions result in a change in biological activity, then moresubstantial changes, denominated “exemplary substitutions” in the Table,or as further described below in reference to amino acid classes, may beintroduced and the products screened.

TABLE of Amino Acid Substitutions Original Exemplary Preferred ResidueSubstitutions Substitutions Ala (A) val; leu; ile val Arg (R) lys; gln;asn lys Asn (N) gln; his; asp, lys; arg gln Asp (D) glu; asn glu Cys (C)ser; ala ser Gln (Q) asn; glu asn Glu (E) asp; gln asp Gly (G) Ala alaHis (H) asn; gln; lys; arg arg Ile (I) leu; val; met; ala; phe;norleucine leu Leu (L) norleucine; ile; val; met; ala; phe ile Lys (K)arg; gln; asn arg Met (M) leu; phe; ile leu Phe (F) leu; val; ile; ala;tyr tyr Pro (P) Ala ala Ser (S) Thr thr Thr (T) Ser ser Trp (W) tyr; phetyr Tyr (Y) trp; phe; thr; ser phe Val (V) ile; leu; met; phe; ala;norleucine leu

Substantial modifications in the biological properties of the antibodyare accomplished by selecting substitutions that differ significantly intheir effect on maintaining (a) the structure of the polypeptidebackbone in the area of the substitution, for example, as a sheet orhelical conformation, (b) the charge or hydrophobicity of the moleculeat the target site, or (c) the bulk of the side chain. Naturallyoccurring residues are divided into groups based on common side-chainproperties:

(1) hydrophobic: norleucine, met, ala, val, leu, ile;

(2) neutral hydrophilic: cys, ser, thr;

(3) acidic: asp, glu;

(4) basic: asn, gln, his, lys, arg;

(5) residues that influence chain orientation: gly, pro: and

(6) aromatic: trp, tyr, phe.

Non-conservative substitutions will entail exchanging a member of one ofthese classes for another class.

Any cysteine residue not involved in maintaining the proper conformationof the anti-CD20 antibody also may be substituted, generally withserine, to improve the oxidative stability of the molecule and preventaberrant crosslinking. Conversely, cysteine bond(s) may be added to theantibody to improve its stability (particularly where the antibody is anantibody fragment such as an Fv fragment).

A particularly preferred type of substitutional variant involvessubstituting one or more hypervariable region residues of a parentantibody (e.g. a humanized or human antibody). Generally, the resultingvariant(s) selected for further development will have improvedbiological properties relative to the parent antibody from which theyare generated. A convenient way for generating such substitutionalvariants involves affinity maturation using phage display. Briefly,several hypervariable region sites (e.g. 6-7 sites) are mutated togenerate all possible amino substitutions at each site. The antibodyvariants thus generated are displayed in a monovalent fashion fromfilamentous phage particles as fusions to the gene III product of M13packaged within each particle. The phage-displayed variants are thenscreened for their biological activity (e.g. binding affinity) as hereindisclosed. In order to identify candidate hypervariable region sites formodification, alanine scanning mutagenesis can be performed to identifyhypervariable region residues contributing significantly to antigenbinding. Alternatively, or additionally, it may be beneficial to analyzea crystal structure of the antigen-antibody complex to identify contactpoints between the antibody and human CD20. Such contact residues andneighboring residues are candidates for substitution according to thetechniques elaborated herein. Once such variants are generated, thepanel of variants is subjected to screening as described herein andantibodies with superior properties in one or more relevant assays maybe selected for further development.

Another type of amino acid variant of the antibody alters the originalglycosylation pattern of the antibody. By altering is meant deleting oneor more carbohydrate moieties found in the antibody, and/or adding oneor more glycosylation sites that are not present in the antibody.

Glycosylation of antibodies is typically either N-linked or O-linked.N-linked refers to the attachment of the carbohydrate moiety to the sidechain of an asparagine residue. The tripeptide sequencesasparagine-X-serine and asparagine-X-threonine, where X is any aminoacid except proline, are the recognition sequences for enzymaticattachment of the carbohydrate moiety to the asparagine side chain.Thus, the presence of either of these tripeptide sequences in apolypeptide creates a potential glycosylation site. O-linkedglycosylation refers to the attachment of one of the sugarsN-aceylgalactosamine, galactose, or xylose to a hydroxyamino acid, mostcommonly serine or threonine, although 5-hydroxyproline or5-hydroxylysine may also be used.

Addition of glycosylation sites to the antibody is convenientlyaccomplished by altering the amino acid sequence such that it containsone or more of the above escribed tripeptide sequences (for N-linkedglycosylation sites). The alteration may also be made by the additionof, or substitution by, one or more serine or threonine residues to thesequence of the original antibody (for O-linked glycosylation sites).

Nucleic acid molecules encoding amino acid sequence variants of the and-CD20 antibody are prepared by a variety of methods known in the art.These methods include, but are not limited to, isolation from a naturalsource (in the case of naturally occurring amino acid sequence variants)or preparation by oligonucleotide-mediated (or site-directed)mutagenesis, PCR mutagenesis, and cassette mutagenesis of an earlierprepared variant or a non-variant version of the anti-CD20 antibody.

It may be desirable to modify the antibody of the invention with respectto effector function, e.g. so as to enhance antigen-dependentcell-mediated cytotoxicity (ADCC) and/or complement dependentcytotoxicity (CDC) of the antibody. This may be achieved by introducingone or more amino acid substitutions in an Fe region of the antibody.Alternatively or additionally, cysteine residue(s) may be introduced inthe Fc region, thereby allowing interchain disulfide bond formation inthis region. The homodimeric antibody thus generated may have improvedinternalization capability and/or increased complement-mediated cellkilling and antibody-dependent cellular cytotoxicity (ADCC). See Caronet al., J. Exp Med. 176:1191-1195 (1992) and Shopes, B. J. Immunol.148:2918-2922 (1992). Homodimeric antibodies with enhanced anti-tumoractivity may also be prepared using heterobifunctional cross-linkers asdescribed in Wolff er al. Cancer Research 53:2560-2565 (1993).Alternatively, an antibody can be engineered which has dual Fe regionsand may thereby have enhanced complement mediated lysis and ADCCcapabilities. See Stevenson et al. Anti-Cancer Drug Design 3:219-230(1989).

To increase the serum half life of the antibody, one may incorporate asalvage receptor binding epitope into the antibody (especially anantibody fragment) as described in U.S. Pat. No. 5,739,277, for example.As used herein, the term “salvage receptor binding epitope” refers to anepitope of the Fe region of an IgG molecule (e.g., IgG₁, IgG₂, IgG₃, orIgG₄) that is responsible for increasing the in vivo serum half-life ofthe IgG molecule.

Other Antibody Modifications

Other modifications of the antibody are contemplated herein. Forexample, the antibody may be linked to one of a variety ofnonproteinaceous polymers, e.g., polyethylene glycol, polypropyleneglycol, polyoxyalkylenes, or copolymers of polyethylene glycol andpolypropylene glycol. The antibody also may be entrapped inmicrocapsules prepared, for example, by coacervation techniques or byinterfacial polymerization (for example, hydroxymethylcellulose orgelatin-microcapsules and poly-(methylmethacylate) microcapsules,respectively), in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules), or in macroemulsions. Such techniques are disclosed inRemington's Pharmaceutical Sciences, 16th edition, Oslo. A., Ed.,(1980).

Screening for Antibodies with the Desired Properties

Antibodies with certain biological characteristics may be selected asdescribed in the Experimental Examples.

The growth inhibitory effects of an anti-CD20 antibody of the inventionmay be assessed by methods known in the art. e.g., using cells whichexpress CD20 either endogenously or following transfection with the CD20gene. For example, tumor cell lines and CD20-transfected cells maytreated with an anti-CD20 monoclonal antibody of the invention atvarious concentrations for a few days (e.g., 2-7) days and stained withcrystal violet or MTT or analyzed by some other colorimetric assay.Another method of measuring proliferation would be by comparing³H-thymidine uptake by the cells treated in the presence or absence ananti-CD20 antibody of the invention. After antibody treatment, the cellsare harvested and the amount of radioactivity incorporated into the DNAquantitated in a scintillation counter. Appropriate positive controlsinclude treatment of a selected cell line with a growth inhibitoryantibody known to inhibit growth of that cell line.

To select for antibodies which induce cell death, loss of membraneintegrity as indicated by, e.g., propidium iodide (PI), trypan blue or7AAD uptake may be assessed relative to control. A PI uptake assay canbe performed in the absence of complement and immune effector cells.CD20-expressing tumor cells are incubated with medium alone or mediumcontaining of the appropriate monoclonal antibody at e.g. about 10μg/ml. The cells are incubated for a 3 day time period. Following eachtreatment, cells are washed and aliquoted into 35 mm strainer-capped12×75 tubes (1 ml per tube, 3 tubes per treatment group) for removal ofcell clumps. Tubes then receive PI (I0 μg/ml). Samples may be analyzedusing a FACSCAN™ flow cytometer and FACSCONVERT™ CellQuest software(Becton Dickinson). Those antibodies which induce statisticallysignificant levels of cell death as determined by PI uptake may beselected as cell death-inducing antibodies.

To screen for antibodies which bind to an epitope on CD20 bound by anantibody of interest, a routine cross-blocking assay such as thatdescribed in Antibodies, A Laboratory Manual, Cold Spring HarborLaboratory, Ed Harlow and David Lane (1988), can be performed. Thisassay can be used to determine if a test antibody binds the same site orepitope as an anti-CD20 antibody of the invention. Alternatively, oradditionally, epitope mapping can be performed by methods known in theart. For example, the antibody sequence can be mutagenized such as byalanine scanning, to identify contact residues. The mutant antibody isinitially tested for binding with polyclonal antibody to ensure properfolding. In a different method, peptides corresponding to differentregions of CD20 can be used in competition assays with the testantibodies or with a test antibody and an antibody with a characterizedor known epitope.

Vectors, Host Cells and Recombinant Methods

The invention also provides an isolated nucleic acid encoding ahumanized CD20 binding antibody, vectors and host cells comprising thenucleic acid, and recombinant techniques for the production of theantibody.

For recombinant production of the antibody, the nucleic acid encoding itis isolated and inserted into a replicable vector for further cloning(amplification of the DNA) or for expression. DNA encoding themonoclonal antibody is readily isolated and sequenced using conventionalprocedures (e.g., by using oligonucleotide probes that are capable ofbinding specifically to genes encoding the heavy and light chains of theantibody). Many vectors are available. The vector components generallyinclude, but are not limited to, one or more of the following: a signalsequence, an origin of replication, one or more marker genes, anenhancer element, a promoter, and a transcription termination sequence.

(i) Signal Sequence Component

The CD20 binding antibody of this invention may be producedrecombinantly not only directly, but also as a fusion polypeptide with aheterologous polypeptide, which is preferably a signal sequence or otherpolypeptide having a specific cleavage site at the N-terminus of themature protein or polypeptide. The heterologous signal sequence selectedpreferably is one that is recognized and processed (i.e., cleaved by asignal peptidase) by the host cell. For prokaryotic host cells that donot recognize and process the native CD20 binding antibody signalsequence, the signal sequence is substituted by a prokaryotic signalsequence selected, for example, from the group of the alkalinephosphatase, penicillinase, lpp, or heat-stable enterotoxin II leaders.For yeast secretion the native signal sequence may be substituted by,e.g., the yeast invertase leader, α factor leader (includingSaccharomyces and Kluyveromyces α-factor leaders), or acid phosphataseleader, the C. albicans glucoamylase leader, or the signal described inWO 90/13646. In mammalian cell expression, mammalian signal sequences aswell as viral secretory leaders, for example, the herpes simplex gDsignal, are available.

The DNA for such precursor region is ligated in reading frame to DNAencoding the CD20 binding antibody.

(ii) Origin of Replication

Both expression and cloning vectors contain a nucleic acid sequence thatenables the vector to replicate in one or more selected host cells.Generally, in cloning vectors this sequence is one that enables thevector to replicate independently of the host chromosomal DNA, andincludes origins of replication or autonomously replicating sequences.Such sequences are well known for a variety of bacteria, yeast, andviruses. The origin of replication from the plasmid pBR322 is suitablefor most Gram-negative bacteria, the 2μ plasmid origin is suitable foryeast, and various viral origins (SV40, polyoma, adenovirus, VSV or BPV)are useful for cloning vectors in mammalian cells. Generally, the originof replication component is not needed for mammalian expression vectors(the SV40 origin may typically be used only because it contains theearly promoter).

(iii) Selection Gene Component

Expression and cloning vectors may contain a selection gene, also termeda selectable marker. Typical selection genes encode proteins that (a)confer resistance to antibiotics or other toxins, e.g., ampicillin,neomycin, methotrexate, or tetracycline, (b) complement auxotrophicdeficiencies, or (c) supply critical nutrients not available fromcomplex media, e.g., the gene encoding D-alanine racemase for Bacilli.

One example of a selection scheme utilizes a drug to arrest growth of ahost cell. Those cells that are successfully transformed with aheterologous gene produce a protein conferring drug resistance and thussurvive the selection regimen. Examples of such dominant selection usethe drugs neomycin, mycophenolic acid and hygromycin.

Another example of suitable selectable markers for mammalian cells arethose that enable the identification of cells competent to take up theCD20 binding antibody nucleic acid, such as DHFR, thymidine kinase,metallothionein-I and -II, preferably primate metallothionein genes,adenosine deaminase, ornithine decarboxylase, etc.

For example, cells transformed with the DHFR selection gene are firstidentified by culturing all of the transformants in a culture mediumthat contains methotrexate (Mtx), a competitive antagonist of DHFR. Anappropriate host cell when wild-type DHFR is employed is the Chinesehamster ovary (CHO) cell line deficient in DHFR activity (e.g., ATCCCRL-9096).

Alternatively, host cells (particularly wild-type hosts that containendogenous DHFR) transformed or co-transformed with DNA sequencesencoding CD20 binding antibody, wild-type DHFR protein, and anotherselectable marker such as aminoglycoside 3-phosphotransferase (APH) canbe selected by cell growth in medium containing a selection agent forthe selectable marker such as an aminoglycosidic antibiotic, e.g.,kanamycin, neomycin, or 0418. See U.S. Pat. No. 4,965,199.

A suitable selection gene for use in yeast is the trp1 gene present inthe yeast plasmid YRp7 (Stinchcomb et al., Nature, 282:39 (1979)). Thetrp1 gene provides a selection marker for a mutant strain of yeastlacking the ability to grow in tryptophan, for example, ATCC No. 44076or PEP4-1. Jones, Genetics, 85:12 (1977). The presence of the trp1lesion in the yeast host cell genome then provides an effectiveenvironment for detecting transformation by growth in the absence oftryptophan. Similarly, Leu2-deficient yeast strains (ATCC 20,622 or38,626) are complemented by known plasmids bearing the Leu2 gene.

In addition, vectors derived from the 1.6 μm circular plasmid pKD1 canbe used for transformation of Kluyveromyces yeasts. Alternatively, anexpression system for large-scale production of recombinant calfchymosin was reported for K. lactis. Van den Berg, Bio/Technology, 8:135(1990). Stable multi-copy expression vectors for secretion of maturerecombinant human serum albumin by industrial strains of Kluyveromyceshave also been disclosed. Fleer et al., Bio/Technology, 9:968-975(1991).

-   -   (iv) Promoter Component

Expression and cloning vectors usually contain a promoter that isrecognized by the host organism and is operably linked to the nucleicacid encoding the CD20 binding antibody. Promoters suitable for use withprokaryotic hosts include the phoA promoter, β-lactamase and lactosepromoter systems, alkaline phosphatase promoter, a tryptophan (trp)promoter system, and hybrid promoters such as the tac promoter. However,other known bacterial promoters are suitable. Promoters for use inbacterial systems also will contain a Shine-Dalgarno (S.D.) sequenceoperably linked to the DNA encoding the CD20 binding antibody.

Promoter sequences are known for eukaryotes. Virtually all eukaryoticgenes have an AT-rich region located approximately 25 to 30 basesupstream from the site where transcription is initiated. Anothersequence found 70 to 80 bases upstream from the start of transcriptionof many genes is a CNCAAT region where N may be any nucleotide. At the3′ end of most eukaryotic genes is an AATAAA sequence that may be thesignal for addition of the poly A tail to the 3 end of the codingsequence. All of these sequences are suitably inserted into eukaryoticexpression vectors.

Examples of suitable promoter sequences for use with yeast hosts includethe promoters for 3-phosphoglycerate kinase or other glycolytic enzymes,such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase,pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphateisomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphateisomerase, phosphoglucose isomerase, and glucokinase.

Other yeast promoters, which are inducible promoters having theadditional advantage of transcription controlled by growth conditions,are the promoter regions for alcohol dehydrogenase 2, isocytochrome C,acid phosphatase, degradative enzymes associated with nitrogenmetabolism, metallothionein, glyceraldehyde-3-phosphate dehydrogenase,and enzymes responsible for maltose and galactose utilization. Suitablevectors and promoters for use in yeast expression are further describedin EP 73,657. Yeast enhancers also are advantageously used with yeastpromoters.

CD20 binding antibody transcription from vectors in mammalian host cellsis controlled, for example, by promoters obtained from the genomes ofviruses such as polyoma virus, fowlpox virus, adenovirus (such asAdenovirus 2), bovine papilloma virus, avian sarcoma virus,cytomegalovirus, a retrovirus, hepatitis-B virus and most preferablySimian Virus 40 (SV40), from heterologous mammalian promoters, e.g., theactin promoter or an immunoglobulin promoter, from heat-shock promoters,provided such promoters are compatible with the host cell systems.

The early and late promoters of the SV40 virus are conveniently obtainedas an SV40 restriction fragment that also contains the SV40 viral originof replication. The immediate early promoter of the humancytomegalovirus is conveniently obtained as a HindIII E restrictionfragment. A system for expressing DNA in mammalian hosts using thebovine papilloma virus as a vector is disclosed in U.S. Pat. No.4,419,446. A modification of this system is described in U.S. Pat. No.4,601,978. See also Reyes er al., Nature 297:598-601 (1982) onexpression of human β-interferon cDNA in mouse cells under the controlof a thymidine kinase promoter from herpes simplex virus. Alternatively,the Rous Sarcoma Virus long terminal repeat can be used as the promoter.

(v) Enhancer Element Component

Transcription of a DNA encoding the CD20 binding antibody of thisinvention by higher eukaryotes is often increased by inserting anenhancer sequence into the vector. Many enhancer sequences are now knownfrom mammalian genes (globin, elastase, albumin. α-fetoprotein, andinsulin). Typically, however, one will use an enhancer from a eukaryoticcell virus. Examples include the SV40 enhancer on the late side of thereplication origin (bp 100-270), the cytomegalovirus early promoterenhancer, the polyoma enhancer on the late side of the replicationorigin, and adenovirus enhancers. See also Yaniv, Nature 297:17-18(1982) on enhancing elements for activation of eukaryotic promoters. Theenhancer may be spliced into the vector at a position 5′ or 3′ to theCD20 binding antibody-encoding sequence, but is preferably located at asite 5′ from the promoter.

(vi) Transcription Termination Component

Expression vectors used in eukaryotic host cells (yeast, fungi, insect,plant, animal, human, or nucleated cells from other multicellularorganisms) will also contain sequences necessary for the termination oftranscription and for stabilizing the mRNA. Such sequences are commonlyavailable from the 5 and, occasionally 3′, untranslated regions ofeukaryotic or viral DNAs or cDNAs. These regions contain nucleotidesegments transcribed as polyadenylated fragments in the untranslatedportion of the mRNA encoding CD20 binding antibody. One usefultranscription termination component is the bovine growth hormonepolyadenylation region. See WO94/11026 and the expression vectordisclosed therein.

(vii) Selection and Transformation of Host Cells

Suitable host cells for cloning or expressing the DNA in the vectorsherein are the prokaryote, yeast, or higher eukaryote cells describedabove. Suitable prokaryotes for this purpose include cubacteria, such asGram-negative or Gram-positive organisms, for example,Enterobacteriaceae such as Escherichia, e.g., E. coli, Enterobacter,Erwinia, Klebsiella, Proteus, Salmonella. e.g., Salmonella typhimurium,Serratia, e.g., Serratia marcescans, and Shigella, as well as Bacillisuch as B. subtilis and B. licheniformis (e.g., B. licheniformis 41Pdisclosed in DD 266,710 published 12 Apr. 1969), Pseudomonas such as P.aeruginosa, and Streptomyces. One preferred E. coli cloning host is E.coli 294 (ATCC 31,446), although other strains such as E. coli B, E.coli X1776 (ATCC 31,537), and E. coli W3110 (ATCC 27,325) are suitable.These examples are illustrative rather than limiting.

Full length antibody, antibody fragments, and antibody fusion proteinscan be produced in bacteria, in particular when glycosylation and Fceffector function are not needed, such as when the therapeutic antibodyis conjugated to a cytotoxic agent (e.g., a toxin) and theimmunoconjugate by itself shows effectiveness in tumor cell destruction.Full length antibodies have greater half life in circulation. Productionin E. coli is faster and more cost efficient. For expression of antibodyfragments and polypeptides in bacteria, see. e.g., U.S. Pat. No.5,648,237 (Carter et. al.), U.S. Pat. No. 5,789,199 (Joly et al.), andU.S. Pat. No. 5,840,523 (Simmons et al.) which describes translationinitiation region (TIR) and signal sequences for optimizing expressionand secretion, these patents incorporated herein by reference. Afterexpression, the antibody is isolated from the E. coli cell paste in asoluble fraction and can be purified through, e.g., a protein A or Gcolumn depending on the isotype. Final purification can be carried outsimilar to the process for purifying antibody expressed e.g., in CHOcells.

In addition to prokaryotes, eukaryotic microbes such as filamentousfungi or yeast are suitable cloning or expression hosts for CD20 bindingantibody-encoding vectors. Saccharomyces cerevisiae, or common baker'syeast, is the most commonly used among lower eukaryotic hostmicroorganisms. However, a number of other genera, species, and strainsare commonly available and useful herein, such as Schizosaccharomycespombe; Kluyveromyces hosts such as, e.g., K. lactis, K. fragilis (ATCC12,424). K. bulgaricus (ATCC 16,045), K. wickeramii (ATCC 24,178). K.waltii (ATCC 56,500), K. drosophilarum (ATCC 36,906), K. thermotolerans,and K. marxianus; yarrowia (EP 402,226); Pichia pastoris (EP 183,070);Candida; Trichoderma reesia (EP 244,234); Neurospora crassa;Schwanniomyces such as Schwanniomyces occidentalis; and filamentousfungi such as, e.g., Neurospora, Penicillium. Tolypocladium, andAspergillus hosts such as A. nidulans and A niger.

Suitable host cells for the expression of glycosylated CD20 bindingantibody are derived from multicellular organisms. Examples ofinvertebrate cells include plant and insect cells. Numerous baculoviralstrains and variants and corresponding permissive insect host cells fromhosts such as Spodoptera frugiperda (caterpillar), Aedes aegypti(mosquito), Aedes albopictus (mosquito), Drosophila melanogaster(fruitfly), and Bombyx mori have been identified. A variety of viralstrains for transfection are publicly available, e.g., the L-1 variantof Autographa californica NPV and the Bm-5 strain of Bombyx mori NPV,and such viruses may be used as the virus herein according to thepresent invention, particularly for transfection of Spodopterafrugiperda cells.

Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato,and tobacco can also be utilized as hosts.

However, interest has been greatest in vertebrate cells, and propagationof vertebrate cells in culture (tissue culture) has become a routineprocedure. Examples of useful mammalian host cell lines are monkeykidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); humanembryonic kidney line (293 or 293 cells subcloned for growth insuspension culture, Graham et al., J. Gen Virol. 36:59 (1977)); babyhamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovarycells/-DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216(1980)); mouse sertoli cells (TM4, Mather, Biol. Reprod 23:243-251(1980)); monkey kidney cells (CV1 ATCC CCL 70); African green monkeykidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells(HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo ratliver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT060562, ATCC CCL51); TRI cells (Mather et al., Annals N.Y. Acad. Sci.383:44-68 (1982)); MRC 5 cells; FS4 cells; and a human hepatoma line(Hep G2).

Host cells are transformed with the above-described expression orcloning vectors for CD20 binding antibody production and cultured inconventional nutrient media modified as appropriate for inducingpromoters, selecting transformants, or amplifying the genes encoding thedesired sequences.

(viii) Culturing the Host Cells

The host cells used to produce the CD20 binding antibody of thisinvention may be cultured in a variety of media. Commercially availablemedia such as Ham's F10 (Sigma), Minimal Essential Medium ((MEM),(Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium((DMEM), Sigma) are suitable for culturing the host cells. In addition,any of the media described in Ham et al., Meth. Enz. 58:44 (1979),Barnes et al., Anal. Biochem. 102:255 (1980), U.S. Pat. Nos. 4,767,704;4,657,866; 4,927,762; 4,560,655; or 5,122,469; WO 90/03430; WO 87/00195;or U.S. Pat. No. Re. 30,985 may be used as culture media for the hostcells. Any of these media may be supplemented as necessary with hormonesand/or other growth factors (such as insulin, transferrin, or epidermalgrowth factor), salts (such as sodium chloride, calcium, magnesium, andphosphate), buffers (such as HEPFS), nucleotides (such as adenosine andthymidine), antibiotics (such as GENTAMYCIN™ drug), trace elements(defined as inorganic compounds usually present at final concentrationsin the micromolar range), and glucose or an equivalent energy source.Any other necessary supplements may also be included at appropriateconcentrations that would be known to those skilled in the art. Theculture conditions, such as temperature, pH, and the like, are thosepreviously used with the host cell selected for expression, and will beapparent to the ordinarily skilled artisan.

(ix) Purification of Antibody

When using recombinant techniques, the antibody can be producedintracellularly, in the periplasmic space, or directly secreted into themedium. If the antibody is produced intracellularly, as a first step,the particulate debris, either host cells or lysed fragments, areremoved, for example, by centrifugation or ultrafitration. Carter etal., Bio/Technology 10:163-167 (1992) describe a procedure for isolatingantibodies which are secreted to the periplasmic space of E. coli.Briefly, cell paste is thawed in the presence of sodium acetate (pH3.5), EDTA, and phenylmethylsulfonylfluoride (PMSF) over about 30 min.Cell debris can be removed by centrifugation. Where the antibody issecreted into the medium, supernatants from such expression systems aregenerally first concentrated using a commercially available proteinconcentration filter, for example, an Amicon or Millipore Pelliconultrafiltration unit. A protease inhibitor such as PMSF may be includedin any of the foregoing steps to inhibit proteolysis and antibiotics maybe included to prevent the growth of adventitious contaminants.

The antibody composition prepared from the cells can be purified using,for example, hydroxylapatite chromatography, gel electrophoresis,dialysis, and affinity chromatography, with affinity chromatographybeing the preferred purification technique. The suitability of protein Aas an affinity ligand depends on the species and isotype of anyimmunoglobulin Fc domain that is present in the antibody. Protein A canbe used to purify antibodies that are based on human γ1, γ2, or γ4 heavychains (Lindmark et al., J. Immunol. Meth. 62:1.13 (1983)). Protein G isrecommended for all mouse isotypes and for human γ3 (Guss et al., EMBOJ. 5:15671575 (1986)). The matrix to which the affinity ligand isattached is most often agarose, but other matrices are available.Mechanically stable matrices such as controlled pore glass orpoly(styrenedivinyl)benzene allow for faster flow rates and shorterprocessing times than can be achieved with agarose. Where the antibodycomprises a C_(H)3 domain, the Bakerhond ABX™ resin (J. T. Baker,Phillipsburg. N.J.) is useful for purification. Other techniques forprotein purification such as fractionation on an ion-exchange column,ethanol precipitation, Reverse Phase HPLC, chromatography on silica,chromatography on heparin SEPHAROSE™ chromatography on an anion orcation exchange resin (such as a polyaspartic acid column),chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation are alsoavailable depending on the antibody to be recovered.

Following any preliminary purification step(s), the mixture comprisingthe antibody of interest and contaminants may be subjected to low pHhydrophobic interaction chromatography using an elution buffer at a pHbetween about 2.5-4.5, preferably performed at low salt concentrations(e.g., from about 0-0.25M salt).

Antibody Conjugates

The antibody may be conjugated to a cytotoxic agent such as a toxin or aradioactive isotope. In certain embodiments, the toxin is calicheamicin,a maytansinoid, a dolastatin, auristatin E and analogs or derivativesthereof, are preferable.

Preferred drugs/toxins include DNA damaging agents, inhibitors ofmicrotubule polymerization or depolymerization and antimetabolites.Preferred classes of cytotoxic agents include, for example, the enzymeinhibitors such as dihydrofolate reductase inhibitors, and thymidylatesynthase inhibitors, DNA intercalators, DNA cleavers, topoisomeraseinhibitors, the anthracycline family of drugs, the vinca drugs, themitomycins, the bleomycins, the cytotoxic nucleosides, the pteridinefamily of drugs, diynenes, the podophyllotoxins and differentiationinducers. Particularly useful members of those classes include, forexample, methotrexate, methopterin, dichloromethotrexate,5-fluorouracil, 6-mercaptopurine, cytosine arabinoside, melphalan,leurosine, leurosideine, actinomycin, daunorubicin, doxorubicin,N-(5,5-diacetoxypentyl)doxorubicin, morpholino-doxorubicin,1-(2-choroehthyl)-1,2-dimethanesulfonyl hydrazide, Ns-acetyl spermidine,aminopterin methopterin, esperamicin, mitomycin C, mitomycin A,actinomycin, bleomycin, carminomycin, aminopterin, tallysomycin,podophyllotoxin and podophyllotoxin derivatives such as etoposide oretoposide phosphate, vinblastine, vincristine, vindesine, taxol,taxotere, retinoic acid, butyric acid, N⁸-acetyl spermidine,camptothecin, calicheamicin, bryostatins, cephalostatins, ansamitocin,actosin, maytansinoids such as DM-1, maytansine, maytansinol,N-desmethyl-4,5-descpoxymaytansinol, C-19-dechloromaytansinol,C-20-hydroxymaytansinol, C-20-demethoxymaytansinol, C-9-SH maytansinol,C-14-alkoxymethylmaytansinol, C-14-hydroxy or acetyloxymethlmaytansinol,C-15-hydroxy/acetyloxymaytansinol, C-15-methoxymaytansinol,C-18-N-demethylmaytansinol and 4,5-deoxymaytansinol, auristatins such asauristatin E, M, PHE and PE; dolostatins such as dolostatin A,dolostatin B, dolostatin C, dolostatin D, dolostatin E (20-epi and11-epi), dolostatin G, dolostatin H1, dolostatin 1, dolostatin 1,dolostatin 2, dolostatin 3, dolostatin 4, dolostatin 5, dolostatin 6,dolostatin 7, dolostatin 8, dolostatin 9, dolostatin 10, deo-dolostatin10, dolostatin 11, dolostatin 12, dolostatin 13, dolostatin 14,dolostatin 15, dolostatin 16, dolostatin 17, and dolostatin 18;cephalostatins such as cephalostatin 1, cephalostatin 2, cephalostatin3, cephalostatin 4, cephalostatin 5, cephalostatin 6, cephalostatin 7,25′-epi-cephalostatin 7, 20-epi-cephalostatin 7, cephalostatin 8,cephalostatin 9, cephalostatin 10, cephalostatin 11, cephalostatin 12,cephalostatin 13, cephalostatin 14, cephalostatin 15, cephalostatin 16,cephalostatin 17, cephalostatin 18, and cephalostatin 19.

Maytansinoids are mitototic inhibitors which act by inhibiting tubulinpolymerization. Maytansine was first isolated from the east Africanshrub Maytenus serrata (U.S. Pat. No. 3,896,111). Subsequently, it wasdiscovered that certain microbes also produce maytansinoids, such asmaytansinol and C-3 maytansinol esters (U.S. Pat. No. 4,151,042).Synthetic maytansinol and derivatives and analogues thereof aredisclosed, for example, in U.S. Pat. Nos. 4,137,230; 4,248,870;4,256,746; 4,260,608; 4,265,814; 4,294,757; 4,307,016; 4,308,268;4,308.269; 4,309,428; 4,313,946; 4,315,929; 4,317,821; 4,322,348;4,331,598; 4,361.650; 4,364,866; 4,424,219; 4,450,254; 4,362,663; and4,371,533, the disclosures of which are hereby expressly incorporated byreference.

Maytansine and maytansinoids have been conjugated to antibodiesspecifically binding to tumor cell antigens. Immunoconjugates containingmaytansinoids and their therapeutic use are disclosed, for example, inU.S. Pat. Nos. 5,208,020, 5,416,064 and European Patent RP 0 425 235 B1,the disclosures of which are hereby expressly incorporated by reference.Liu et al., Proc. Natl. Acad. Sci. USA 93:8618-8623 (1996) describedimmunoconjugates comprising a maytansinoid designated DM1 linked to themonoclonal antibody C242 directed against human colorectal cancer. Theconjugate was found to be highly cytotoxic towards cultured colon cancercells, and showed antitumor activity in an in vivo tumor growth assay.Chari et al., Cancer Research 2:127-131 (1992) describe immunoconjugatesin which a maytansinoid was conjugated via a disulfide linker to themurine antibody A7 binding to an antigen on human colon cancer celllines, or to another murine monoclonal antibody TA.1 that binds theHER-2/neu oncogene.

There are many linking groups known in the art for makingantibody-maytansinoid conjugates, including, for example, thosedisclosed in U.S. Pat. No. 5,208,020 or EP Patent 0 425 235 B1, andChari e al., Cancer Research 52: 127-131 (1992). The linking groupsinclude disulfide groups, thioether groups, acid labile groups,photolabile groups, peptidase labile groups, or esterase labile groups,as disclosed in the above-identified patents, disulfide and thioethergroups being preferred.

Conjugates of the antibody and maytansinoid may be made using a varietyof bifunctional protein coupling agents such asN-succinimidyl-3-(2-pyridyldithio) propionate (SPDP),succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate,iminothiolane (IT), bifunctional derivatives of imidoesters (such asdimethyl adipimidate HCL), active esters (such as disuccinimidylsuberate), aldehydes (such as glutareldehyde), bis-azido compounds (suchas bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (suchas bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astoluene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). Particularly preferred coupling agentsinclude N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP) (Carlssonet al., Biochem. J. 173:723-737 [1978]) andN-succinimidyl-4-(2-pyridylthio)pentanoate (SPP) to provide for adisulfide linkage.

The linker may be attached to the maytansinoid molecule at variouspositions, depending on the type of the link. For example, an esterlinkage may be formed by reaction with a hydroxyl group usingconventional coupling techniques. The reaction may occur at the C-3position having a hydroxyl group, the C-14 position modified withhyrdoxymethyl, the C-15 position modified with a hydroxyl group, and theC-position having a hydroxyl group. In a preferred embodiment, thelinkage is formed at the C-3 position of maytansinol or a maytansinolanalogue.

Calicheamicin

Another immunoconjugate of interest comprises an CD20 binding antibodyconjugated to one or more calicheamicin molecules. The calicheamicinfamily of antibiotics are capable of producing double-stranded DNAbreaks at sub-picomolar concentrations. For the preparation ofconjugates of the calicheamicin family, see U.S. patents 5,712.374,5,714,586, 5,739,116, 5,767,285, 5,770,701, 5,770,710, 5,773,001,5,877,296 (all to American Cyanamid Company). Structural analogues ofcalicheamicin which may be used include, but are not limited to, γ₁^(I), α₂ ^(I), α₃ ^(I). N-acetyl-γ₁ ^(I), PSAG and 2^(I) ₁, (Hinman etal. Cancer Research 53: 3336-3342 (1993), Lode et al. Cancer Research58: 2925.2928 (1998) and the aforementioned U.S. patents to AmericanCyanamid). Another anti-tumor drug that the antibody can be conjugatedis QFA which is an antifolate. Both calicheamicin and QFA haveintracellular sites of action and do not readily cross the plasmamembrane. Therefore, cellular uptake of these agents through antibodymediated internalization greatly enhances their cytotoxic effects.

Radioactive Isotopes

For selective destruction of the tumor, the antibody may comprise ahighly radioactive atom. A variety of radioactive isotopes are availablefor the production of radioconjugated anti-CD20 antibodies. Examplesinclude At¹¹¹, I¹³³, I¹²⁵, Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³, Bi²¹², P³², Pb²¹²and radioactive isotopes of Lu. When the conjugate is used fordiagnosis, it may comprise a radioactive atom for scintigraphic studies,for example tc^(99m) or I¹²³, or a spin label for nuclear magneticresonance (NMR) imaging (also known as magnetic resonance imaging, mri),such as iodine-123 again, iodine-131, indium-111, fluorine-19,carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese or iron.

The radio- or other labels may be incorporated in the conjugate in knownways. For example, the peptide may be biosynthesized or may besynthesized by chemical amino acid synthesis using suitable amino acidprecursors involving, for example, fluorine-19 in place of hydrogen.Labels such as tc^(99m) or I¹²³, Re¹⁸⁶, Re¹⁸⁸ and In¹¹¹ can be attachedvia a cysteine residue in the peptide. Yttrium-90 can be attached via alysine residue. The IODOGEN method (Fraker et al (1978) Biochem.Biophys. Res. Commun. 80: 49-57 can be used to incorporate iodine-123.“Monoclonal Antibodies in Immunoscintigraphy” (Chatal, CRC Press 1989)describes other methods in detail.

Conjugates of the antibody and cytotoxic agent may be made using avariety of bifunctional protein coupling agents such asN-succinimidyl-3-(2-pyridyldithio) propionate (SPDP),succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate,iminothiolane (IT), bifunctional derivatives of imidoesters (such asdimethyl adipimidate HCL), active esters (such as disuccinimidylsuberate), aldehydes (such as glutareldehyde), his-azido compounds (suchas bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (suchas bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin canbe prepared as described in Vitetta et al. Science 238: 1098 (1987).Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent forconjugation of radionucleotide to the antibody. See WO94/11026. Thelinker may be a “cleavable linker” facilitating release of the cytotoxicdrug in the cell. For example, an acid-labile linker,peptidase-sensitive linker, photolabile linker, dimethyl linker ordisulfide-containing linker (Chari et al., Cancer Research 52: 127-131(1992); U.S. Pat. No. 5,208,020) may be used.

Therapeutic Uses of the CD20 Binding Antibodies

The CD20 binding antibodies of the invention are useful to treat anumber of malignant and non-malignant diseases including autoimmunediseases and related conditions, and CD20 positive cancers including Bcell lymphomas and leukemias. Stem cells (B-cell progenitors) in bonemarrow lack the CD20 antigen, allowing healthy B-cells to regenerateafter treatment and return to normal levels within several months.

Autoimmune diseases or autoimmune related conditions include arthritis(rheumatoid arthritis, juvenile rheumatoid arthritis, osteoarthritis,psoriatic arthritis), psoriasis, dermatitis including atopic dermatitis;chronic autoimmune urticaria, polymyositis/dermatomyositis, toxicepidermal necrolysis, systemic scleroderma and sclerosis, responsesassociated with inflammatory bowel disease (IBD) (Crohn's disease,ulcerative colitis), respiratory distress syndrome, adult respiratorydistress syndrome (ARDS), meningitis, allergic rhinitis, encephalitis,uveitis, colitis, glomerulonephritis, allergic conditions, eczema,asthma, conditions involving infiltration of T cells and chronicinflammatory responses, atherosclerosis, autoimmune myocarditis,leukocyte adhesion deficiency, systemic lupus erythematosus (SLE), lupus(including nephritis, non-renal, discoid, alopecia), juvenile onsetdiabetes, multiple sclerosis, allergic encephalomyelitis, immuneresponses associated with acute and delayed hypersensitivity mediated bycytokines and T-lymphocytes, tubercukosis, sarcoidosis, granulomatosisincluding Wegener's granulomatosis, agranulocytosis, vasculitis(including ANCA), aplastic anemia, Coombs positive anemia. DiamondBlackfan anemia, immune hemolytic anemia including autoimmune hemolyticanemia (AIHA), pernicious anemia, pure red cell aplasia (PRCA), FactorVIII deficiency, hemophilia A, autoimmune neutropenia, pancytopenia,leukopenia, diseases involving leukocyte diapedesis, CNS inflammatorydisorders, multiple organ injury syndrome, myasthenia gravis,antigen-antibody complex mediated diseases, anti-glomerular basementmembrane disease, anti-phospholipid antibody syndrome, allergicneuritis, Bechet disease, Castleman's syndrome, Goodpasture's Syndrome,Lambert-Eaton Myasthenic Syndrome, Reynaud's syndrome, Sjorgen'ssyndrome, Stevens-Johnson syndrome, solid organ transplant rejection(including pretreatment for high panel reactive antibody titers, IgAdeposit in tissues, etc), graft versus host disease (GVHD), pemphigoidbullous, pemphigus (all including vulgaris, foliaceus), autoimmunepolyendocrinopathies, Reiter's disease, stiff-man syndrome, giant cellarteritis, immune complex nephritis, IgA nephropathy, IgMpolyneuropathies or IgM mediated neuropathy, idiopathic thrombocytopenicpurpura (ITP), thrombotic throbocytopenic purpura (TTP), autoimmunethrombocytopenia, autoimmune disease of the testis and ovary includingautoimmune orchitis and oophoritis, primary hypothyroidism; autoimmuneendocrine diseases including autoimmune thyroiditis, chronic thyroiditis(Hashimoto's Thyroiditis), subacute thyroiditis, idiopathichypothyroidism, Addison's disease, Grave's disease, autoimmunepolyglandular syndromes (or polyglandular endocrinopathy syndromes),Type I diabetes also referred to as insulin-dependent diabetes mellitus(IDDM) and Sheehan's syndrome; autoimmune hepatitis, Lymphoidinterstitial pneumonitis (HIV), bronchiolitis obliterans(non-transplant) vs NSIP, Guillain-Barre' Syndrome, Large VesselVasculitis (including Polymyalgia Rheumatica and Giant Cell (Takayasu's)Arteritis), Medium Vessel Vasculitis (including Kawasaki's Disease andPolyarteritis Nodosa), ankylosing spondylitis. Berger's Disease (IgAnephropathy), Rapidly Progressive Glomerulonephritis, Primary biliarycirrhosis, Celiac sprue (gluten enteropathy), Cryoglobulinemia, ALS,coronary artery disease.

CD20 positive cancers are those comprising abnormal proliferation ofcells that express CD20 on the cell surface. The CD20 positive B cellneoplasms include CD20-positive Hodgkin's disease including lymphocytepredominant Hodgkin's disease (LPHD); non-Hodgkin's lymphoma (NHL);follicular center cell (FCC) lymphomas; acute lymphocytic leukemia(ALL); chronic lymphocytic leukemia (CLL); Hairy cell leukemia. Thenon-Hodgkins lymphoma include low grade/follicular non-Hodgkin'slymphoma (NHL), small lymphocytic lymphoma (SLL), intermediategrade/follicular NHL, intermediate grade diffuse NHL, high gradeimmunoblastic NHL, high grade lymphoblastic NHL, high grade smallnon-cleaved cell NHL, bulky disease NHL, plasmacytoid lymphocyticlymphoma, mantle cell lymphoma. AIDS-related lymphoma and Waldenstrom'smacroglobulinemia. Treatment of relapses of these cancers are alsocontemplated. LPHD is a type of Hodgkin's disease that tends to relapsefrequently despite radiation or chemotherapy treatment and ischaracterized by CD20-positive malignant cells. CLL is one of four majortypes of leukemia. A cancer of mature B-cells called lymphocytes, CLL ismanifested by progressive accumulation of cells in blood, bone marrowand lymphatic tissues.

In specific embodiments, the humanized CD20 binding antibodies andfunctional fragments thereof are used to treat non-Hodgkin's lymphoma(NHL), lymphocyte predominant Hodgkin's disease (LPHD), smalllymphocytic lymphoma (SLL), chronic lymphocytic leukemia, rheumatoidarthritis and juvenile rheumatoid arthritis, systemic lupuserythematosus (SLE) including lupus nephritis. Wegener's disease,inflammatory bowel disease, idiopathic thrombocytopenic purpura (ITP),thrombotic throbocytopenic purpura (TTP), autoimmune thrombocytopenia,multiple sclerosis, psoriasis, IgA nephropathy, IgM polyneuropathies,myasthenia gravis, vasculitis, diabetes mellitus. Reynaud's syndrome,Sjorgen's syndrome and glomerulonephritis.

The humanized CD20 binding antibodies or functional fragments thereofare useful as a single-agent treatment in, e.g., for relapsed orrefractory low-grade or follicular, CD20-positive, B-cell NHL, or can beadministered to patients in conjunction with other drugs in a multi drugregimen.

Indolent lymphoma is a slow-growing, incurable disease in which theaverage patient survives between six and 10 years following numerousperiods of remission and relapse. In one embodiment, the humanized CD20binding antibodies or functional fragments thereof are used to treatindolent NHL.

The parameters for assessing efficacy or success of treatment of theneoplasm will be known to the physician of skill in the appropriatedisease. Generally, the physician of skill will look for reduction inthe signs and symptoms of the specific disease. Parameters can includemedian time to disease progression, time in remission, stable disease.

The following references describe lymphomas and CLL, their diagnoses,treatment and standard medical procedures for measuring treatmentefficacy. Canellos G P, Lister, T A, Sklar J L: The Lymphomas. W.B.Saunders Company, Philadelphia, 1998; van Besien K and Cabanillas, F:Clinical Manifestations, Staging and Treatment of Non-Hodgkin'sLymphoma, Chap. 70, pp 1293-1338, in: Hematology, Basic Principles andPractice, 3rd ed. Hoffman et al. (editors). Churchill Livingstone,Philadelphia, 2000; and Rai, K and Patel, D: Chronic LymphocyticLeukemia, Chap. 72, pp 1350-1362, in: Hematology. Basic Principles andPractice, 3rd ed. Hoffman et al. (editors). Churchill Livingstone,Philadelphia, 2000.

The parameters for assessing efficacy or success of treatment of anautoimmune or autoimmune related disease will be known to the physicianof skill in the appropriate disease. Generally, the physician of skillwill look for reduction in the signs and symptoms of the specificdisease. The following are by way of examples.

In one embodiment, the antibodies of the invention are useful to treatrheumatoid arthritis. RA is characterized by inflammation of multiplejoints, cartilage loss and bone erosion that leads to joint destructionand ultimately reduced joint function. Additionally, since RA is asystemic disease, it can have effects in other tissues such as thelungs, eyes and bone marrow. Fewer than 50 percent of patients who havehad RA for more than 10 years can continue to work or function normallyon a day-to-day basis.

The antibodies can be used as first-line therapy in patients with earlyRA (i.e., methotrexate (MTX) naive) and as monotherapy, or incombination with, e.g., MTX or cyclophosphamide. Or, the antibodies canbe used in treatment as second-line therapy for patients who were DMARDand/or MTX refractory, and as monotherapy or in combination with, e.g.,MTX. The humanized CD20 binding antibodies are useful to prevent andcontrol joint damage, delay structural damage, decrease pain associatedwith inflammation in RA, and generally reduce the signs and symptoms inmoderate to severe RA. The RA patient can be treated with the humanizedCD20 antibody prior to, after or together with treatment with otherdrugs used in treating RA (see combination therapy below). In oneembodiment, patients who had previously failed disease-modifyingantirheumatic drugs and/or had an inadequate response to methotrexatealone are treated with a humanized CD20 binding antibody of theinvention. In one embodiment of this treatment, the patients are in a17-day treatment regimen receiving humanized CD20 binding antibody alone(Ig iv infusions on days 1 and 15); CD20 binding antibody pluscyclophosphamide (750 mg iv infusion days 3 and 17); or CD20 bindingantibody plus methotrexate.

One method of evaluating treatment efficacy in RA is based on AmericanCollege of Rheumatology (ACR) criteria, which measures the percentage ofimprovement in tender and swollen joints, among other things. The RApatient can be scored at for example, ACR 20 (20 percent improvement)compared with no antibody treatment (e.g., baseline before treatment) ortreatment with placebo. Other ways of evaluating the efficacy ofantibody treatment include X-ray scoring such as the Sharp X-ray scoreused to score structural damage such as bone erosion and joint spacenarrowing. Patients can also be evaluated for the prevention of orimprovement in disability based on Health Assessment Questionnaire [HAQ]score, AIMS score, SF-36 at time periods during or after treatment. TheACR 20 criteria may include 20% improvement in both tender (painful)joint count and swollen joint count plus a 20% improvement in at least 3of 5 additional measures:

-   -   1. patient's pain assessment by visual analog scale (VAS),    -   2. patient's global assessment of disease activity (VAS),    -   3. physician's global assessment of disease activity (VAS),    -   4. patient's self-assessed disability measured by the Health        Assessment Questionnaire, and    -   5. acute phase reactants, CRP or ESR.        The ACR 50 and 70 are defined analogously. Preferably, the        patient is administered an amount of a CD20 binding antibody of        the invention effective to achieve at least a score of ACR 20,        preferably at least ACR 30, more preferably at least ACR50, even        more preferably at least ACR70, most preferably at least ACR 75        and higher.

Psoriatic arthritis has unique and distinct radiographic features. Forpsoriatic arthritis, joint erosion and joint space narrowing can beevaluated by the Sharp score as well. The humanized CD20 bindingantibodies of the invention can be used to prevent the joint damage aswell as reduce disease signs and symptoms of the disorder.

Yet another aspect of the invention is a method of treating Lupus orSIF, by administering to the patient suffering from SLE, atherapeutically effective amount of a humanized CD20 binding antibody ofthe invention. SLEDAI scores provide a numerical quantitation of diseaseactivity. The SLEDAI is a weighted index of 24 clinical and laboratoryparameters known to correlate with disease activity, with a numericalrange of 0-103, see Bryan Gescuk & John Davis, “Novel therapeutic agentfor systemic lupus erythematosus” in Current Opinion in Rheumatology2002, 14:515-521. Antibodies to double-stranded DNA are believed tocause renal flares and other manifestations of lupus. Patientsundergoing antibody treatment can be monitored for time to renal flare,which is defined as a significant, reproducible increase in serumcreatinine, urine protein or blood in the urine. Alternatively or inaddition, patients can be monitored for levels of antinuclear antibodiesand antibodies to double-stranded DNA. Treatments for SLE includehigh-dose corticosteroids and/or cyclophosphamide (HDCC).

Spondyloarthropathies am a group of disorders of the joints, includingankylosing spondylitis, psoriatic arthritis and Crohn's disease.Treatment success can be determined by validated patient and physicianglobal assessment measuring tools.

Various medications are used to treat psoriasis; treatment differsdirectly in relation to disease severity. Patients with a more mild formof psoriasis typically utilize topical treatments, such as topicalsteroids, anthralin, calcipotriene, clobetasol, and tazarotene, tomanage the disease while patients with moderate and severe psoriasis aremore likely to employ systemic (methotrexate, retinoids, cyclosporine.PUVA and UVB) therapies. Tars are also used. These therapies have acombination of safety concerns, time consuming regimens, or inconvenientprocesses of treatment. Furthermore, some require expensive equipmentand dedicated space in the office setting. Systemic medications canproduce serious side effects, including hypertension, hyperlipidemia,bone marrow suppression, liver disease, kidney disease andgastrointestinal upset. Also, the use of phototherapy can increase theincidence of skin cancers. In addition to the inconvenience anddiscomfort associated with the use of topical therapies, phototherapyand systemic treatments require cycling patients on and off therapy andmonitoring lifetime exposure due to their side effects.

Treatment efficacy for psoriasis is assessed by monitoring changes inclinical signs and symptoms of the disease including Physician's GlobalAssessment (PGA) changes and Psoriasis Area and Severity Index (PASI)scores, Psoriasis Symptom Assessment (PSA), compared with the baselinecondition. The patient can be measured periodically throughout treatmenton the Visual analog scale used to indicate the degree of itchingexperienced at specific time points.

Patients may experience an infusion reaction or infusion-relatedsymptoms with their first infusion of a therapeutic antibody. Thesesymptoms vary in severity and generally are reversible with medicalintervention. These symptoms include but are not limited to, flu-likefever, chills/rigors, nausea, urticaria, headache, bronchospasm,angioedema. It would be desirable for the disease treatment methods ofthe present invention to minimize infusion reactions. Thus, anotheraspect of the invention is a method of treating the diseases disclosedby administering a humanized CD20 binding antibody wherein the antibodyhas reduced or no complement dependent cytotoxicity and results inreduced infusion related symptoms as compared to treatment withRituxan®. In one embodiment, the humanized CD20 binding antibody is2H7.v116.

Dosage

Depending on the indication to be treated and factors relevant to thedosing that a physician of skill in the field would be familiar with,the antibodies of the invention will be administered at a dosage that isefficacious for the treatment of that indication while minimizingtoxicity and side effects. For the treatment of a CD20 positive canceror an autoimmune disease, the therapeutically effective dosage will bein the range of about 250 mg/m² to about 400 mg/m² or 500 mg/m2,preferably about 250-375 mg/m². In one embodiment, the dosage range is275-375 mg/m². In one embodiment of the treatment of a CD20 positive Bcell neoplasm, the antibody is administered at a range of 300.375 mg/m².For the treatment of patients suffering from B-cell lymphoma such asnon-Hodgkins lymphoma, in a specific embodiment, the anti-CD20antibodies and humanized anti-CD20 antibodies of the invention will beadministered to a human patient at a dosage of 10 mg/kg or 375 mg/m².For treating NHL, one dosing regimen would be to administer one dose ofthe antibody composition a dosage of 10 mg/kg in the first week oftreatment, followed by a 2 week interval, then a second dose of the sameamount of antibody is administered. Generally, NHL patients receive suchtreatment once during a year but upon recurrence of the lymphoma, suchtreatment can be repeated. In another dosing regimen, patients treatedwith low-grade NHL receive four weeks of a version of humanized 2H7,preferably v16 (375 mg/m2 weekly) followed at week five by threeadditional courses of the antibody plus standard CHOP (cyclophosphamide,doxorubicin, vincristine and prednisone) or CVP (cyclophosphamide,vincristine, prednisone) chemotherapy, which was given every three weeksfor three cycles.

For treating rheumatoid arthritis, in one embodiment, the dosage rangefor the humanized antibody is 125 mg/m² (equivalent to about 200mg/dose) to 600 mg/m², given in two doses, e.g., the first dose of 200mg is administered on day one followed by a second dose of 200 mg on day15. In different embodiments, the dosage is 250 mg/dose, 275 mg, 300 mg,325 mg, 350 mg, 375 mg, 400 mg, 425 mg, 450 mg, 475 mg, 500 mg, 525 mg,550 mg, 575 mg, 600 mg.

In treating disease, the CD20 binding antibodies of the invention can beadministered to the patient chronically or intermittently, as determinedby the physician of skill in the disease.

A patient administered a drug by intravenous infusion or subcutaneouslymay experience adverse events such as fever, chills, burning sensation,asthenia and headache. To alleviate or minimize such adverse events, thepatient may receive an initial conditioning dose(s) of the antibodyfollowed by a therapeutic dose. The conditioning dose(s) will be lowerthan the therapeutic dose to condition the patient to tolerate higherdosages.

Route of Administration

The CD20 binding antibodies are administered to a human patient inaccord with known methods, such as by intravenous administration, e.g.,as a bolus or by continuous infusion over a period of time, bysubcutaneous, intramuscular, intraperitoneal, intracerobrospinal,intra-articular, intrasynovial, intrathecal, or inhalation routes,generally by intravenous or subcutaneous administration.

In on embodiment, the humanized 2H7 antibody is administered byintravenous infusion with 0.9% sodium chloride solution as an infusionvehicle.

Combination Therapy

In treating the B cell neoplasms described above, the patient can betreated with the CD20 binding antibodies of the present invention inconjunction with one or more therapeutic agents such as achemotherapeutic agent in a multidrug regimen. The CD20 binding antibodycan be administered concurrently, sequentially, or alternating with thechemotherapeutic agent, or after non-responsiveness with other therapy.Standard chemotherapy for lymphoma treatment may includecyclophosphamide, cytarabine, melphalan and mitoxantrone plus melphalan.CHOP is one of the most common chemotherapy regimens for treatingNon-Hodgkin's lymphoma. The following are the drugs used in the CHOPregimen: cyclophosphamide (brand names cytoxan, neosar); adriamycin(doxorubicin/hydroxydoxorubicin); vincristine (Oncovin); andprednisolone (sometimes called Deltasone or Orasone). In particularembodiments, the CD20 binding antibody is administered to a patient inneed thereof in combination with one or more of the followingchemotherapeutic agents of doxorubicin, cyclophosphamide, vincristineand prednisolone. In a specific embodiment, a patient suffering from alymphoma (such as a non-Hodgkin's lymphoma) is treated with an anti-CD20antibody of the present invention in conjunction with CHOP(cyclophosphamide, doxorubicin, vincristine and prednisone) therapy. Inanother embodiment, the cancer patient can be treated with a humanizedCD20 binding antibody of the invention in combination with CVP(cyclophosphamide, vincristine, and prednisone) chemotherapy. In aspecific embodiment, the patient suffering from CD20-positive NHL istreated with humanized 2H7.v16 in conjunction with CVP. In a specificembodiment of the treatment of CLL, the CD20 binding antibody isadministered in conjunction with chemotherapy with one or both offludarabine and cytoxan.

In treating the autoimmune diseases or autoimmune related conditionsdescribed above, the patient can be treated with the CD20 bindingantibodies of the present invention in conjunction with a secondtherapeutic agent, such as an immunosuppressive agent, such as in amulti drug regimen. The CD20 binding antibody can be administeredconcurrently, sequentially or alternating with the immunosuppressiveagent or upon non-responsiveness with other therapy. Theimmunosuppressive agent can be administered at the same or lesserdosages than as set forth in the art. The preferred adjunctimmunosuppressive agent will depend on many factors, including the typeof disorder being treated as well as the patient's history.

“Immunosuppressive agent” as used herein for adjunct therapy refers tosubstances that act to suppress or mask the immune system of a patient.Such agents would include substances that suppress cytokine production,down regulate or suppress self-antigen expression, or mask the MHCantigens. Examples of such agents include steroids such asglucocorticosteroids, e.g., prednisone, methylprednisolone, anddexamethasone; 2-amino-6-aryl-5-substituted pyrimidines (see U.S. Pat.No. 4,665,077), azathioprine (or cyclophosphamide, if there is anadverse reaction to azathioprine); bromocryptine; glutaraldehyde (whichmasks the MHC antigens, as described in U.S. Pat. No. 4,120,649):anti-idiotypic antibodies for MHC antigens and MHC fragments;cyclosporin A; cytokine or cytokine receptor antagonists includinganti-interferon-α, -β, or -γ antibodies; anti-tumor necrosis factor-αantibodies; anti-tumor necrosis factor-β antibodies; anti-interleukin-2antibodies and anti-IL-2 receptor antibodies; anti-L3T4 antibodies;heterologous anti-lymphocyte globulin; pan-T antibodies, preferablyanti-CD3 or anti-CD4/CD4a antibodies; soluble peptide containing a LFA-3binding domain (WO 90M08187 published Jul. 26, 1990); streptokinase;TGF-β; streptodornase; RNA or DNA from the host; FK506; RS-61443;deoxyspergualin; rapamycin; T-cell receptor (U.S. Pat. No. 5,114,721);T-cell receptor fragments (Offner et al., Science 251:430-432 (1991); WO90/11294; and WO 91/01133); and T cell receptor antibodies (EP 340,109)such as T10B9.

For the treatment of rheumatoid arthritis, the patient can be treatedwith a CD20 antibody of the invention in conjunction with any one ormore of the following drugs: DMARDS (disease-modifying anti-rheumaticdrugs (e.g., methotrexate), NSAI or NSAID (non-steroidalanti-inflammatory drugs), HUMIRA™ (adalimumab; Abbott Laboratories),ARAVA® (leflunomide), REMICADE® (infliximab; Centocor Inc., of Malvern,Pa.), ENBREL (etanercept; Immunex, Wash.), COX-2 inhibitors. DMARDscommonly used in RA are hydroxycloroquine, sulfasalazine, methotrexate,leflunomide, etanercept, infliximab, azathioprine, D-penicillamine, Gold(oral), Gold (intramuscular), minocycline, cyclosporine, Staphylococcalprotein A immunoadsorption. Adalimumab is a human monoclonal antibodythat hinds to TNFα. Infliximab is a chimeric monoclonal antibody thatbinds to TNFα. Etanercept is an “immunoadhesin” fusion proteinconsisting of the extracellular ligand binding portion of the human 75kD (p75) tumor necrosis factor receptor (TNFR) linked to the Fc portionof a human IgG1. For conventional treatment of RA, see, e.g.,“Guidelines for the management of rheumatoid arthritis” Arthritis &Rheumatism 46(2): 328-346 (February, 2002). In a specific embodiment,the RA patient is treated with a CD20 antibody of the invention inconjunction with methotrexate (MTX). An exemplary dosage of MTX is about7.5-25 mg/kg/wk. MTX can be administered orally and subcutaneously.

For the treatment of ankylosing spondylitis, psoriatic arthritis andCrohn's disease, the patient can be treated with a CD20 binding antibodyof the invention in conjunction with, for example, Remicade®(infliximab; from Centocor Inc., of Malvern, Pa.), ENBREL (etanercept:Immunex, Wash.).

Treatments for SLE include high-dose corticosteroids and/orcyclophosphamide (HDCC). For the treatment of psoriasis, patients can beadministered a CD20 binding antibody in conjunction with topicaltreatments, such as topical steroids, anthralin, calcipotriene,clobetasol, and tazarotene, or with methotrexate, retinoids,cyclosporine, PUVA and UVB therapies. In one embodiment, the psoriasispatient is treated with the CD20 binding antibody sequentially orconcurrently with cyclosporine.

Pharmaceutical Formulations

Therapeutic formulations of the CD20-binding antibodies used inaccordance with the present invention are prepared for storage by mixingan antibody having the desired degree of purity with optionalpharmaceutically acceptable carriers, excipients or stabilizers(Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)),in the form of lyophilized formulations or aqueous solutions. Acceptablecarriers, excipients, or stabilizers are nontoxic to recipients at thedosages and concentrations employed, and include buffers such asphosphate, citrate, and other organic acids; antioxidants includingascorbic acid and methionine; preservatives (such asoctadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride, benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens such as methyl or propyl paraben; catechol;resorcinol: cyclohexanol; 3-pentanol; and m-cresol): low molecularweight (less than about 10 residues) polypeptides; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such asolyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine,histidine, arginine, or lysine; monosaccharides, disaccharides, andother carbohydrates including glucose, mannose, or dextrins; chelatingagents such as EDTA; sugars such as sucrose, mannitol, trehalose orsorbitol; salt-forming counter-ions such as sodium; metal complexes(e.g. Zn-protein complexes); and/or non-ionic surfactants such asTWEEN™, PLURONICS™ or polyethylene glycol (PEG).

Exemplary anti-CD20 antibody formulations are described in WO98/56418,expressly incorporated herein by reference. Another formulation is aliquid multidose formulation comprising the anti-CD20 antibody at 40mg/mL, 25 mM acetate, 150 mM trehalose, 0.9% benzyl alcohol, 0.02%polysorbate 20 at pH 5.0 that has a minimum shelf life of two yearsstorage at 2-8° C. Another anti-CD20 formulation of interest comprises10 mg/mL antibody in 9.0 mg/mL sodium chloride, 7.35 mg/mL, sodiumcitrate dihydrate, 0.7 mg/mL polysorbate 80, and Sterile Water forInjection, pH 6.5. Yet another aqueous pharmaceutical formulationcomprises 10-30 mM sodium acetate from about pH 4.8 to about pH 5.5,preferably at pH5.5, polysorbate as a surfactant in a an amount of about0.01-0.1% v/v, trehalose at an amount of about 2-10% w/v, and benzylalcohol as a preservative (U.S. Pat. No. 6,171,586). Lyophilizedformulations adapted for subcutaneous administration are described inWO97/04801. Such lyophilized formulations may be reconstituted with asuitable diluent to a high protein concentration and the reconstitutedformulation may be administered subcutaneously to the mammal to betreated herein.

One formulation for the humanized 2H7 variants is antibody at 12-14mg/mL in 10 mM histidine, 6% sucrose, 0.02% polysorbate 20, pH 5.8.

In a specific embodiment, 2H7 variants and in particular 2H7.v16 isformulated at 20 mg/mL antibody in 10 mM histidine sulfate, 60 mg/mlsucrose., 0.2 mg/ml polysorbate 20, and Sterile Water for Injection, atpH5.8.

The formulation herein may also contain more than one active compound asnecessary for the particular indication being treated, preferably thosewith complementary activities that do not adversely affect each other.For example, it may be desirable to further provide a cytotoxic agent,chemotherapeutic agent, cytokine or immunosuppressive agent (e.g. onewhich acts on T cells, such as cyclosporin or an antibody that binds Tcells, e.g. one which binds LFA-1). The effective amount of such otheragents depends on the amount of antibody present in the formulation, thetype of disease or disorder or treatment, and other factors discussedabove. These are generally used in the same dosages and withadministration routes as described herein or about from 1 to 99% of theheretofore employed dosages.

The active ingredients may also be entrapped in microcapsules prepared,for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsules and poly-(methylmethacylate) microcapsules,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules) or in macroemulsions. Such techniques are disclosed inRemington's Pharmaceutical Sciences 16th edition. Osol, A. Ed. (1980).

Sustained-release preparations may be prepared. Suitable examples ofsustained-release preparations include semi-permeable matrices of solidhydrophobic polymers containing the antagonist, which matrices are inthe form of shaped articles, e.g. films, or microcapsules. Examples ofsustained-release matrices include polyesters, hydrogels (for example,poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides(U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid andethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradablelactic acid-glycolic acid copolymers such as the LUPRON DEPOT™(injectable microspheres composed of lactic acid-glycolic acid copolymerand leuprolide acetate), and poly-D-(−)-3-hydroxybutyric acid.

The formulations to be used for in vivo administration must be sterile.This is readily accomplished by filtration through sterile filtrationmembranes.

Articles of Manufacture and Kits

Another embodiment of the invention is an article of manufacturecontaining materials useful for the treatment of autoimmune diseases andrelated conditions and CD20 positive cancers such as non-Hodgkin'slymphoma. The article of manufacture comprises a container and a labelor package insert on or associated with the container. Suitablecontainers include, for example, bottles, vials, syringes, etc. Thecontainers may be formed from a variety of materials such as glass orplastic. The container holds a composition which is effective fortreating the condition and may have a sterile access port (for examplethe container may be an intravenous solution bag or a vial having astopper pierceable by a hypodermic injection needle). At least oneactive agent in the composition is a CD20 binding antibody of theinvention. The label or package insert indicates that the composition isused for treating the particular condition. The label or package insertwill further comprise instructions for administering the antibodycomposition to the patient. Package insert refers to instructionscustomarily included in commercial packages of therapeutic products,that contain information about the indications, usage, dosage,administration, contraindications and/or warnings concerning the use ofsuch therapeutic products. In one embodiment, the package insertindicates that the composition is used for treating non-Hodgkins'lymphoma.

Additionally, the article of manufacture may further comprise a secondcontainer comprising a pharmaceutically-acceptable buffer, such asbacteriostatic water for injection (BWFI), phosphate-buffered saline,Ringer's solution and dextrose solution. It may further include othermaterials desirable from a commercial and user standpoint, includingother buffers, diluents, filters, needles, and syringes.

Kits are also provided that are useful for various purposes, e.g., forB-cell killing assays, as a positive control for apoptosis assays, forpurification or immunoprecipitation of CD20 from cells. For isolationand purification of CD20, the kit can contain an anti-CD20 antibodycoupled to heads (e.g., sepharose beads). Kits can be provided whichcontain the antibodies for detection and quantitation of CD20 in vitro,e.g. in an ELISA or a Western blot. As with the article of manufacture,the kit comprises a container and a label or package insert on orassociated with the container. The container holds a compositioncomprising at least one anti-CD20 antibody of the invention. Additionalcontainers may be included that contain, e.g., diluents and buffers,control antibodies. The label or package insert may provide adescription of the composition as well as instructions for the intendedin vitro or diagnostic use.

Cynomolgus Monkey CD20

The invention also provides an isolated nucleic acid comprising thenucleotide sequence of SEQ ID NO.: 24 of the Cynomolgus monkey CD20 asshown in FIG. 19. In one embodiment, the nucleic acid is a cDNA. In oneembodiment, the nucleic acid encoding the monkey CD20 is in anexpression vector for expression in a host cell. The nucleotide sequenceof SEQ ID NO.: 24 in the expression vector is operably linked to anexpression control sequence such as a promoter or promoter and enhancer.The expression control sequence can be can be the native sequencenormally associated with the Cynomolgus CD20 gene, or heterologous tothe gene. Also provided is an isolated polypeptide comprising the aminoacid sequence [SEQ ID NO. 25; FIGS. 19 & 20] of the Cynomolgus monkeyCD20, as well as host cells containing the Cynomolgus CD20 nucleic acid.In one aspect the host cells are eukaryotic cells. e.g., CHO cells.Fusion proteins comprising the Cynomolgus CD20 amino acid sequence orfragments of the sequence are also contemplated.

EXPERIMENTAL EXAMPLES Example 1 Humanization of 2H7 Anti-CD20 MurineMonoclonal Antibody

Humanization of the murine anti-human CD20 antibody, 2H7 (also referredto herein as m2H7, m for murine), was carried out in a series ofsite-directed mutagenesis steps. The murine 2H7 antibody variable regionsequences and the chimeric 2H7 with the mouse V and human C have beendescribed, see. e.g., U.S. Pat. Nos. 5,846,818 and 6,204,023. The CDRresidues of 2H7 were identified by comparing the amino acid sequence ofthe murine 2H7 variable domains (disclosed in U.S. Pat. No. 5,846,818)with the sequences of known antibodies (Kabat et al., Sequences ofproteins of immunological interest, Ed. 5. Public Health Service,National Institutes of Health. Bethesda. Md. (1991)). The CDR regionsfor the light and heavy chains were defined based on sequencehypervariability (Kabat et al., supra) and are shown in FIG. 1A and FIG.1B, respectively. Using synthetic oligonucleotides (Table 1),site-directed mutagenesis (Kunkel, Proc. Natl. Acad. Sci. 82:488-492(1985)) was used to introduce all six of the murine 2H17 CDR regionsinto a complete human Fab framework corresponding to a consensussequence V_(κ)I_(H)III (V_(L) kappa subgroup 1, V_(H) subgroup III)contained on plasmid pVX4 (FIG. 2).

The phagemid pVX4 (FIG. 2) was used for mutagenesis as well as forexpression of F(ab)s in E. coli. Based on the phagemid pb0720, aderivative of pB0475 (Cunningham et al., Science 243: 1330-1336 (1989)),pVX4 contains a DNA fragment encoding a humanized consensus κ-subgroup Ilight chain (V_(L)κI-C_(L)) and a humanized consensus subgroup III heavychain (V_(H)III-C_(H)1) anti-IFN-α (interferon α) antibody, pVX4 alsohas an alkaline phosphatase promotor and Shine-Dalgamo sequence bothderived from another previously described pUC119-based plasmid, pAK2(Carter et al., Proc. Natl. Acad. Sci. USA 89: 4285 (1992)). A uniqueSpel restriction site was introduced between the DNA encoding for theF(ab) light and heavy chains. The first 23 amino acids in bothanti-IFN-α heavy and light chains are the SrII secretion signal sequence(Chang et al., Gene 55: 189-196 (1987)).

To construct the CDR-swap version of 2H7 (2H7.v₂), site-directedmutagenesis was performed on a deoxyuridine-containing template of pVX4;all six CDRs of anti-IFN-α were changed to the murine 2H7 CDRs. Theresulting molecule is referred to as humanized 2H7 version 2 (2H7.v2),or the “CDR-swap version” of 2H7; it has the m2H7 CDR residues with theconsensus human FR residues shown in FIGS. 1A and 1B. Humanized 2H7.v2was used for further humanization.

Table 1 shows the oligonucleotide sequence used to create each of themurine 2H7 (m2H7) CDRs in the H and L chain. For example, the CDR-H1oligonucleotide was used to recreate the m217 H chain CDR1. CDR-H1,CDR-112 and CDR-H3 refers to the H chain CDR1, CDR2 and CDR3,respectively; similarly. CDR-L1. CDR-L2 and CDR-L3 refers to each of theL chain CDRs. The substitutions in CDR-H2 were done in two steps withtwo oligonucleotides, CDR-H2A and CDR-H2B.

TABLE 1 Oligonucleotide sequences used forconstruction of the CDR-swap of murine2H7 CDRs into a human framework in pVX4.Residues changed by each oligonucleotide are underlined. OligonucleotideSubstitution sequence CDR-H1 C TAG ACC TTC ACG AGC TATAAC ATG CAC TGG GTC CG (SEQ ID NO. 27) CDR-H2A G ATT AAT CCT GAC AAC GGCGAC ACG AGC TAT AAC CAG AAG TTC AAG GGC CG (SEQ ID NO. 28) CDR-H2BGAA TGG GTT GCA GCG ATC TAT CCT GGC AAC GGC GAC AC (SEQ ID NO. 29)CDR-H3 AT TAT TGT GCT CGA GTG GTC TAC TAT AGC AAC AGC TAC TGGTAC TTC GAC GTC TGG GGT CAA GGA (SEQ ID NO. 30) CDR-L1C TGC ACA GCC AGC TCT TCT GTC AGC TAT ATG CAT TG (SEQ ID NO. 31) CDR-L2AA CTA CTG ATT TAC GCT CCA TCG AAC CTC GCG TCT GGA GTC C (SEQ ID NO. 32)CDR-L3 TAT TAC TGT CAA CAG TGG AGC TTC AAT CCG CCC ACA TTT GGACAG (SEQ ID NO. 33)

For comparison with humanized constructs, a plasmid expressing achimeric 2H7 Fab (containing marine V_(L) and V_(H) domains, and humanC_(L) and CH₁ domains) was constructed by site-directed mutagenesis(Kunkel, supra) using synthetic oligonucleotides to introduce the murineframework residues into 2H7.v2. The sequence of the resulting plasmidconstruct for expression of the chimeric Fab known as 2H7.v6.8, is shownin FIG. 3. Each encoded chain of the Fab has a 23 amino acid Sillsecretion signal sequence as described for pVX4 (FIG. 2) above.

Based on a sequence comparison of the murine 217 framework residues withthe human V_(κ)I, V_(H)III consensus framework (FIGS. 1A and 1B) andpreviously humanized antibodies (Carter et al., Proc. Natl. Acad. Sci.USA 89:4285-4289 (1992)), several framework mutations were introducedinto the 2H7.v2 Fab construct by site-directed mutagenesis. Thesemutations result in a change of certain human consensus frameworkresidues to those found in the murine 2H7 framework, at sites that mightaffect CDR conformations or antigen contacts. Version 3 containedVH(R71V, N73K), version 4 contained VH(R71V), version 5 containedV_(H)(R71V, N73K) and V_(L)(I46P), and version 6 contained VH(R71V,N73K) and V_(L)(L46P, L47W).

Humanized and chimeric Fab versions of m2H7 antibody were expressed inE. coli and purified as follows. Plasmids were transformed into E. colistrain XL-1 Blue (Stratagene, San Diego, Calif.) for preparation ofdouble- and single-stranded DNA. For each variant, both light and heavychains were completely sequenced using the dideoxynucleotide method(Sequenase, U.S. Biochemical Corp.). Plasmids were transformed into E.coli strain 16C9, a derivative of MM294, plated onto LB platescontaining 5 μg/ml carbenicillin, and a single colony selected forprotein expression. The single colony was grown in 5 ml LB-100 μg/mlcarbenicillin for 5-8 h at 37° C. The 5 ml culture was added to 500 mlAPS-100 μg/ml carbenicillin and allowed to grow for 16 h in a 4 Lbaffled shake flask at 37° C. APS media consists of: 1.5 g glucose, 11.0Hycase SF, 0.6 g yeast extract (certified), 0.19 g anhydrous MgSO₄, 1.07g NH₄Cl, 3.73 g KCl, 1.2 g NaCl, 120 ml 1 M triethanolamine, pH 7.4, to1 L water and then sterile filtered through 0.1 μm Sealkeen filter.

Cells were harvested by centrifugation in a 1 L centrifuge bottle(Nalgene) at 3000×g and the supernatant removed. After freezing for 1 h,the pellet was resuspended in 25 ml cold 10 mM MES-10 mM EDTA, pH 5.0(buffer A), 250 μl of 0.1M PMSF (Sigma) was added to inhibit proteolysisand 3.5 ml of stock 10 mg/ml hen egg white lysozyme (Sigma) was added toaid lysis of the bacterial cell wall. After gentle shaking on ice for 1h, the sample was centrifuged at 40,000×g for 15 min. The supernatantwas brought to 50 ml with buffer A and loaded onto a 2 ml DEAE columnequilibrated with buffer A. The flow-through was then applied to aprotein G-Sepharose CL-4B (Pharmacia) column (0.5 ml bed volume)equilibrated with buffer A. The column was washed with 10 ml buffer Aand eluted with 3 ml 0.3 M glycine, pH 3.0, into 1.25 ml 1 M Tris, pH8.0. The F(ab) was then buffer exchanged into PBS using a Centricon-30(Amicon) and concentrated to a final volume of 0.5 ml. SDS-PAGE gels ofall F(ab)s were run to ascertain purity and the molecular weight of eachvariant was verified by electrospray mass spectrometry.

In cell-based ELISA binding assays (described below), the binding ofFabs, including chimeric 2H7 Fab, to CD20 was difficult to detect.Therefore, the 2H7 Fab versions were reformatted as full-length IgG1antibodies for assays and further mutagenesis.

Plasmids for expression of full-length IgG's were constructed bysubcloning the V_(L) and V_(H) domains of chimeric 2H7 (v6.8) Fab aswell as humanized Fab versions 2 to 6 into previously described pRKvectors for mammalian cell expression (Gorman et al., DNA Prot. Eng.Tech. 2:3-10 (1990)). Briefly, each Fab construct was digested withEcoRV and BlpI to excise a V_(L) fragment, which was cloned into theEcoRV/BlpI sites of plasmid pDR1 (FIG. 4) for expression of the completelight chain (V_(L)-C_(L) domains). Additionally, each Fab construct wasdigested with PvuII and ApaI to excise a V_(H) fragment, which wascloned into the PvuII/ApaI sites of plasmid pDR2 (FIG. 5) for expressionof the complete heavy chain (VH-CH₁-hinge-CH₂—CH₃ domains). For each IgGvariant, transient transfections were performed by cotransfecting alight-chain expressing plasmid and a heavy-chain expressing plasmid intoan adenovirus-transformed human embryonic kidney cell line, 293 (Grahamet al., J. Gen. Virol., 36:59-74, (1977)). Briefly, 293 cells were spliton the day prior to transfection, and plated in serum-containing medium.On the following day, double-stranded DNA prepared as a calciumphosphate precipitate was added, followed by pAdVAntage™ DNA (Promega,Madison, Wis.), and cells were incubated overnight at 37° C. Cells werecultured in scrum-free medium and harvested after 4 days. Antibodieswere purified from culture supernatants using protein A-Sepharose CL-4B,then buffer exchanged into 10 mM sodium succinate, 140 mM NaCl, pH 6.0,and concentrated using a Centricon-10 (Amicon). Protein concentrationswere determined by quantitative amino acid analysis.

To measure relative binding affinities to the CD20 antigen, a cell-basedELISA assay was developed. Human B-lymphoblastoid WIL2-S cells (ATCC CRL8885, American Type Culture Collection, Rockville, Md.) were grown inRPMI 1640 supplemented with 2 mM L-glutamine, 20 mM HEPES, pH 7.2 and10% heat-inactivated fetal bovine serum in a humidified 5% CO₂incubator. The cells were washed with PBS containing 1% FBS (assaybuffer) and seeded at 250-300,000 cell/well in 96-well round bottomplates (Nunc, Roskilde, Denmark). Two-fold serially diluted standard(15.6-1000 ng/ml of 2H7 v6.8 chimeric IgG) and threefold seriallydiluted samples (2.7-2000 ng/ml) in assay buffer were added to theplates. The plates were buried in ice and incubated for 45 min. Toremove the unbound antibody, 0.1 mL assay buffer were added to thewells. Plates were centrifuged and supernatants were removed. Cells werewashed two more times with 0.2 mL assay buffer. Antibody bound to theplates was detected by adding peroxidase conjugated goat anti-human Fcantibody (Jackson ImmunoResearch, West Grove, Pa.) to the plates. Aftera 45 min incubation, cells were washed as described before. TMBsubstrate (3,3′,5,5′-tetramethyl benzidine; Kirkegaard & PerryLaboratories, Gaithersburg, Md.) was added to the plates. The reactionwas stopped by adding 1 M phosphoric acid. Titration curves were fitwith a four-parameter nonlinear regression curve-fitting program(KaleidaGraph, Synergy software, Reading, Pa.). The absorbance at themidpoint of the titration curve (mid-OD) and its correspondingconcentration of the standard were determined. Then the concentration ofeach variant at this mid-OD was determined, and the concentration of thestandard was divided by that of each variant. Hence the values are aratio of the binding of each variant relative to the standard. Standarddeviations in relative affinity (equivalent concentration) weregenerally +/−10% between experiments.

As shown in Table 2, binding of the CDR-swap variant (v.2) was extremelyreduced compared to chimeric 2H7 (v.6.8). However, versions 3 to 6showed improved binding. To determine the minimum number of mutationsthat might be required to restore binding affinity to that of chimeric2H7, additional mutations and combinations of mutations were constructedby site-direct mutagenesis to produce variants 7 to 17 as indicated inTable 3. In particular, these included Vx mutations A49G, F67A, 169L,N73K., and L78A; and V_(L) mutations M4L, M331, and F71Y. Versions 16and 17 showed the best relative binding affinities, within 2-fold ofthat of the chimeric version, with no significant difference(s.d.=+/−10%) between the two. To minimize the number of mutations,version 16, having only 4 mutations of human framework residues tomurine framework residues (Table 3), was therefore chosen as thehumanized form for additional characterization.

TABLE 2 Relative binding affinity of humanized2H7 IgG variants to CD20 compared to chimeric2H7 using cell-based ELISA. The relativebinding is expressed as the concentrationof the chimeric 2H7 over the concentrationof the variant required for equivalentbinding; hence a ratio <1 indicatesweaker affinity for the variant. Standarddeviation in relative affinity determinationaveraged +/−10%. Framework substitutionsin the variable domains are relative tothe CDR-swap version according to tire numbering system of Kabat(Kabat et al., supra). Heavy Light chain Chain 2H7 (V_(H)) (V_(L))Relative version substitutions substitutions binding 6.8 (Chimera)(Chimera) −1− 2 (CDR (CDR 0.01 swap) swap) 3 R71V, (CDR 0.21 N73K swap)4 R71V (CDR 0.21 swap) 5 R71V, L46P 0.50 N73K 6 R71V, L46P, 0.58 N73KL47W 7 R71V L46P 0.33 8 R71V, L46P 0.19 L78A 9 R71V, L46P 0.07 F67A 10R71V, L46P 0.12 F67A, I69L 11 R71V, L46P 0.19 F67A, L78A 12 R71V L46P,0.32 M4L 13 R71V L46P, 0.31 M33I 14 R71V L46P, 0.25 F71Y 15 R71V L46P,0.26 M4L, M33I 16 R71V, L46P 0.65 N73K, A49G 17 R71V, L46P, 0.67 N73K,L47W A49G

TABLE 3 Oligonucleotide sequences used forconstruction of mutations VH(A49G, R71V, N73K) and VL(L46P) in humanized2H7 version 16 (2H7.v16). Underlinedcodons encode the indicated amino acidsubstitutions. For V_(H) (R71V, N73K) andV_(L) (L46P), the oligos are shown as thesense strand since these were used formutagenesis on the Fab template, whilefor V_(H) (A49G), the oligo is shown asthe anti-sense strand, since this wasused with the pRK (IgG heavy chain) template. The protein sequence ofversion 16 is shown in FIG. 6 and FIG. 7. Oligonucleotide Substitutionsequence V_(H) GT TTC ACT ATA AGT (R71V, GTC GAC AAG TCC N73K)AAA AAC ACA TT (SEQ ID NO. 34) V_(H) GCCAGGATAGATGGCGC (A49G)CAACCCATTCCAGGCC (SEQ ID NO. 35) AAGCTCCGAAACCACT V_(L) GATTTACGCT(L46P) (SEQ ID NO. 36)

Example 2 Antigen-Binding Determinants (Paratope) of 2H7

Alanine substitutions (Cunningham & Wells. Science 244:1081-1085 (1989)were made in 2H7.v16 or 2H7.v17 in order to test the contributions ofindividual side chains of the antibody in binding to CD20. ISO variantswere expressed in 293 cells from pDR1 and pDR2 vectors, purified, andassayed for relative binding affinity as described above. Severalalanine substitutions resulted In significant decreases in relativebinding to CD20 on WII-2S cells (Table 4).

TABLE 4 Effects of alanine substitutions in the CDR regions of humanized2H7.v16 measured using cell-based ELISA (WIL2-S cells). The relativebinding is expressed as the concentration of the 2H7.v16 parent over theconcentration of the variant required for equivalent binding; hence aratio <1 indicates weaker affinity for the variant; a ratio >1 indicateshigher affinity for the variant. Standard deviation in relative affinitydetermination averaged +/−10%. Framework substitutions in the variabledomains are relative to 2H7.v16 according to the numbering system ofKabat (Kabat et al., supra). 2H7 CDR Heavy chain Light chain Relativeversion location substitutions substitutions binding 16 — — — -1-   140H1 G26A — 0.63 141 H1 Y27A — 0.47 34 H1 T28A — 0.86 35 H1 F29A — 0.07 36H1 T30A — 0.81 37 H1 S31A — 0.97 142 H1 Y32A — 0.63 143 H1 N33A — NDB144 H1 M34A — 1.2  145 H1 H35A — <0.25  146 H2 A50G — 0.31 147 H2 I51A —0.65 38 H2 Y52A — 0.01 148 H2 P52aA — 0.66 39 H2 G53A — 0.89 67 H2 N54A— 1.4  40 H2 G55A — 0.79 41 H2 D56A — 2.0  89 H2 T57A — 0.61 90 H2 S58A— 0.92 91 H2 Y59A — 0.74 92 H2 N60A — 0.80 93 H2 Q61A — 0.83 94 H2 K62A— 0.44 95 H2 F63A — 0.51 83 H2 V71A — 0.96 149 H2 K64A — 0.82 150 H2G65A — 1.2  153 H3 V95A — 0.89 42 H3 V96A — 0.98 43 H3 Y97A — 0.63 44 H3Y98A — 0.40 45 H3 S99A — 0.84; 0.92 46 H3 N100A — 0.81 47 H3 S100aA —0.85 48 H3 Y100bA — 0.78 49 H3 W100cA — 0.02 59 H3 Y100dA — 0.98 60 H3F100eA — NDB 61 H3 D101A — 0.31 151 H3 V102A — 1.1  117 L1 — R24A 0.85118 L1 — A25G 0.86 119 L1 — S26A 0.98 120 L1 — S27A 0.98 121 L1 — S28A1.0 122 L1 — V29A 0.41 50 L1 — S30A 0.96 51 L1 — Y32A 1.0  123 L1 — M33A1.0  124 L1 — H34A 0.21 125 L2 — A50G 0.92 126 L2 — P51A 0.88 52 L2 —S52A 0.80 53 L2 — N53A 0.76 54 L2 — L54A 0.60 127 L2 — A55G 1.1  128 L2— S56A 1.1  129 L3 — Q89A 0.46 130 L3 — Q90A <0.22  55 L2 — W91A 0.88 56L3 — S92A 1.1  57 L3 — F93A 0.36 58 L3 — N94A 0.61 131 L3 — P95A NDB 132L3 — P96A 0.18 133 L3 — T97A <0.22  NBD means no detectable binding. Thetwo numbers for version 45 are from separate experiments.

Example 3 Additional Mutations within 2H7 CDR Regions

Substitutions of additional residues and combinations of substitutionsat CDR positions that were identified as Important by Ala-scanning werealso tested. Several combination variants, particularly v.96 appeared tobind more tightly than v.16.

TABLE 5 Effects of combinations of mutations and non-alaninesubstitutions in the CDR regions of humanized 2H7.v16 measured usingcell-based ELISA (WIL2-S cells). The relative binding to CD20 isexpressed as the concentration of the 2H7.v16 parent over theconcentration of the variant required for equivalent binding; hence aratio <1 indicates weaker affinity for the variant; a ratio >1 indicateshigher affinity for the variant. Standard deviation in relative affinitydetermination averaged +/−10%. Framework substitutions in the variabledomains are relative to 2H7.v16 accordingt o the numbering system ofKabat (Kabat et al., supra). 2H7 Heavy chain Light chain Relativeversion substitutions substitutions binding 16 — — -1-   96 D56A, N100AS92A 3.5  97 S99T, N100G, — 0.99 Y100bI 98 S99G, N100S, — 1.6  Y100bI 99N100G, Y100bI — 0.80 101 N54S, D56A — 1.7  102 N54K, D56A — 0.48 103D56A, N100A — 2.1  104 S99T, N100G — 0.81 105 S99G, N100S — 1.1  106N100G — ~1    167 S100aG, Y100bS — 136 D56A, N100A S56A, S92A 2.6  137D56A, N100A A55G, S92A 2.1  156 D56A, N100A S26A, S56A, 2.1  S92A 107D56A, N100A, S92A not expressed Y100bI 182 Y27W — 183 Y27F — 184 F29Y —185 F29W — 186 Y32F — 187 Y32W — 188 N33Q — 189 N33D — 190 N33Y — 191N33S — 208 H35S — 209 A50S — 210 A50R — 211 A50V — 212 A50L — 168 Y52W —169 Y52F — 0.75 170 N54D — 0.25 171 N54S — 1.2  172 D56K — 1   173 D56R— 174 D56H — 1.5  175 D56E — 1.2  213 D56S — 214 D56G — 215 D56N — 216D56Y — 176 Y59W — 177 Y59F — 180 K62R — 181 K62D — 178 F63W — 179 F63Y —157 Y97W — 0.64 158 Y97F — 1.2  159 Y98W — 0.64 160 Y98F — 0.88 106N100G — 161 W100cY — 0.05 162 W100cF — 0.27 163 F100eY — 0.59 164 F100eW— 0.71 165 D101N — 0.64 166 S99G, N100G, — 0.99 S100aD, Y100b deleted217 V102Y — 1.0  207 — H34Y 192 — Q89E 193 — Q89N 194 — Q90E 195 — Q90N196 — W91Y 197 — W91F 205 — S92N 206 — S92G 198 — F93Y 199 — F93W 204 —F93S, N94Y 200 — P96L 201 — P96Y 202 — P96W 203 — P96R

Example 4 Mutations at Sites of Framework Humanization Substitutions

Substitutions of additional residues at framework positions that werechanged during humanization were also tested in the 21H7.v16 background.In particular, alternative framework substitutions that ware neitherfound in the murine 2H7 parent nor the human consensus framework weremade at V_(L)(P46) and V_(H)(G49, V71, and K73).

The substitutions generally led to little change in relative binding(Table 6), indicating that there is some flexibility in frameworkresidues at these positions.

TABLE 6 Relative binding in a cell-based (WIL2-S) assay of frameworksubstitutions. IgG variants are shown with mutations with respect to the2H7.v16 background. The relative binding is expressed as theconcentration of the 2H7.v6.8 chimera over the concentration of thevariant required for equivalent binding; hence a ratio <1 indicatesweaker affinity for the variant; a ratio >1 indicates higher affinityfor the variant. Standard deviation in relative affinity determinationaveraged +/−10%. Framework substitutions in the variable domains arerelative to 2H7.v16 according to the numbering system of Kabat (Kabat etal., supra). 2H7 Heavy chain Light chain Relative version substitutionssubstitutions binding 6.8 (chimera) (chimera) -1-   16 — — 0.64 78 K73R— 0.72 79 K73H — 0.49 80 K73Q — 0.58 81 V71I — 0.42 82 V71T — 0.58 83V71A — 84 G49S — 0.32 85 G49L — 86 — P46E 0.22 87 — P46V 0.51 88 — P46T108 G49A, V71T, S92A, M32L,  0.026* K73R P46T 109 G49A, A49G, S92A,M32L,  0.026* V71T, K73R P46T 110 K73R, D56A, S92A, M32L Not expressedN100A 111 G49A, V71T, —  0.46* K73R 112 G49A, A50G, —  0.12* V71T, K73R*Variants that were assayed with 2H7.v16 as the standard comparator;relative values are normalized to that of the chimera.

Example 5 Humanized 2H17 Variants with Enhanced Effector Functions

Because 2H7 can mediate lysis of B-cells through bothcomplement-dependent cytotoxicity (CDC) and antibody-dependent cellularcytotoxicity (ADCC), we sought to produce variants of humanized 2H7.v16with improved CDC and ADCC activity. Mutations of certain residueswithin the Fc regions of other antibodies have been described (Idusogieet al., J. Immunol. 106:2571-2575 (2001)) for improving CDC throughenhanced binding to the complement component C1q. Mutations have alsobeen described (Shields et al., J. Biol. Chem. 276:6591-6604 (2001);Presta et al., Biochem. Soc. Trans. 30:487-490 (2002)) for improvingADCC through enhanced IgG binding to activating Fey receptors andreduced IgG binding to inhibitory Fcγ receptors. In particular, threemutations have been identified for improving CDC and ADCC activity.S298A/E333A/K334A (also referred to herein as a triple Ala mutant orvariant; numbering in the Fc region is according to the EU numberingsystem; Kabat et al., supra) as described (Idusogie et al., supra(2001); Shields et al., supra).

In order to enhance CDC and ADCC activity of 2H7, a triple Ala mutant ofthe 2H7 Fc was constructed. A humanized variant of the anti-HER2antibody 4d5 has been produced with mutations S298A/11333A/K334A and isknown as 4D5Fc110 (i.e., anti-p¹⁸⁵HER2 IgG1 (S298A/E333A/K334A); Shieldset al., supra). A plasmid, p4DSFc110 encoding antibody 4DSFc110 (Shieldset al., supra) was digested with ApaI and HindIII, and the Fc-fragment(containing mutations S298A/E333A/K334A) was ligated into theApaI/HindIII sites of the 2H7 heavy-chain vector pDR2-v16, to producepDR2-v31. The amino acid sequence of the version 31 complete H chain isshown in FIG. 8. The L chain is the same as that of v16.

Although the constant domains of the Fc region of IgG1 antibodies armrelatively conserved within a given species, allelic variations exist(reviewed by Lefranc and Lefranc, in The human IgG subclasses: molecularanalysis of structure, function, and regulation, pp. 43-78, F. Shakib(ed.), Pergammon Press, Oxford (1990)).

TABLE 7 Effects of substitutions in the Fc region on CD20 binding.Relative binding to CD20 was measured in a cell-based (WIL2-S) assay offramework substitutions. 2H7 Fc Relative version Substitutions* binding6.8 — -1-   16 — 0.65 31 S298A, E333A, 0.62 K334A Fc mutations * areindicated by EU numbering (Kabat, supra) and are relative to the 2H7.v16parent. The combination of three Ala changes in the Fc region of v.31 isdescribed as “Fc110.” IgG variants are shown with mutations with respectto the 2H7.v16 background. The relative binding is expressed as theconcentration of the 2H7.v6.8 chimera over the concentration of thevariant required for equivalent binding; hence a ratio <1 indicatesweaker affinity for the variant. Standard deviation in relative affinitydetermination averaged +/−10%.

Example 6 Humanized 2H7 Variants with Enhanced Stability

For development as therapeutic proteins, it is desirable to choosevariants that remain stable with respect to oxidation, deamidation, orother processes that may affect product quality, in a suitableformulation buffer. In 2H7.v16, several residues were identified aspossible sources of instability. VL (M32) and VH (M34, N100). Therefore,mutations were introduced at these sites for comparison with v16.

TABLE 8 Relative binding of 2H7 variants designed for enhancedstablility and/or effector function, to CD20 in a cell-based (WIL2-S)assay. IgG variants are shown with mutations with respect to the 2H7.v16background. The relative binding is expressed as the concentration ofthe 2H7.v6.8 chimera over the concentration of the variant required forequivalent binding; hence a ratio <1 indicates weaker affinity for thevariant. Standard deviation in relative affinity determination averaged+/−10%. Heavy Light 2H7 chain chain Fc Relative version (V_(H)) changes(V_(L)) changes changes* binding 6.8 (chimera) (chimera) — -1-   16 — —— 0.65 62 — M32I — 0.46 63 M34I — — 0.49 64 N100A — — 65 N100A L47W —0.74 66 S99A L47W — 0.62 67 N54A — — 68 — M32I — 0.48 69 — M32L — 0.5270 N100A — S298A, E333A, 0.80 K334A 71 N100D — S298A, E333A, 0.44 K334A72 N100A M32I — 0.58 73 N100A M32L — 0.53 74 N100A M32I S298A, E333A,0.61 K334A 75 N100A M32L S298A, E333A, 0.60 K334A 113 — — E356D, M358L 0.60** 114 D56A, N100A M32L, S92A S298A, E333A,  1.2** K334A 115 D56A,N100A M32L, S92A S298A, E333A,  1.4** K334A, E356D, M358L 116 D56A,N100A M32L, S92A S298A, K334A,  1.2** K322A 134 D56A, N100A M32L, S92AE356D, M358L,  1.5** D265A 135 D56A, N100A M32L, S92A E356D, M358L, 0.95** D265A, K326W 138 D56A, N100A M32L, S92A S298A, E333A,  1.2**K334A, K326A 139 D56A, N100A M32L, S92A S298A, E333A,  1.1** K334A,K326A, E356N, M358L 154 — — D265A  0.70** 155 — — S298A, K322A,  0.70**K334A Framework substitutions in the variable domains are relative to2H7.v16 according to the numbering system of Kabat and Fc mutations *are indicated by EU numbering (Kabat et al., supra). **Variants thatwere measured with 2H7.v16 as the standard comparator; relative bindingvalues are normalized to that of the chimera. Additional Fc mutationswere combined with stability or affinity-enhancing mutatuions to alteror enhance effector functions based on previously reported mutations(Idusogie et al. (2000); Idusogie et al. (2001); Shields et al. (2001)).These changes include S298, E333A, K334A as described in Example 5;K322A to reduced CDC activity; D365A to reduce ADCC activity; K326A orK326W to enhance CDC activity; and E356D/M358L to test the effects ofallotypic changes in the Fc region. None of these mutations causedsignificant differences in CD20 binding affinity.

To test the effects of stability mutations on the rate of proteindegradation, 2H7.v16 and 2H7.v73 were formulated at 12-14 mg/mL in 10 mMhistidine, 6% sucrose, 0.02% polysorbate 20, pH 5.8 and incubated at 40°C. for 16 days. The incubated samples were then assayed for changes incharge variants by ion exchange chromatography, aggregation andfragmentation by size exclusion chromatography, and relative binding bytesting in a cell-based (WIL2-S) assay.

The results (FIG. 9) show that 2H7 v.73 has greater stability comparedto 2H7 v.16 with respect to losses in the fraction of main peak by ionexchange chromatography under accelerated stability conditions. Nosignificant differences were seen with respect to aggregation,fragmentation, or binding affinity.

Example 7 Scatchard Analysis of Antibody Binding to CD20 on WIL2-S Cells

Equilibrium dissociation constants (K_(d)) were determined for 2H7 IgGvariants binding to WIL2-S cells using radiolabeled 2H7 IgG. IgGvariants were produced in CHO cells. Rituxan® (source for allexperiments is Genentech, S. San Francisco, Calif.) and murine 2H7 (HDPharMingen, San Diego, Calif.) were used for comparison with humanizedvariants. The murine 2H7 antibody is also available from other sources,e.g., eBioscience, and Calbiochem (both of San Diego, Calif.). AccurateChemical & Scientific Corp., (Westbury, N.Y.). Ancell (Bayport. Minn.),and Vinci-Biochem (Vinci, Italy). All dilutions were performed inbinding assay buffer (DMEM media containing 1% bovine serum albumin, 25mM HEPES pH 7.2, and 0.01% sodium azide). Aliquots (0.025 mL) of¹²⁵I-2H7.v16 (iodinated with lactoperoxidase) at a concentration of 0.8nM were dispensed into wells of a V-bottom 96-well microassay plate, andserial dilutions (0.05 mL) of cold antibody were added and mixed. WIL2-Scells (60,000 cells in 0.025 mL) were then added. The plate was sealedand incubated at room temperature for 24 h, then centrifuged for 15 minat 3,500 RPM. The supernatant was then aspirated and the cell pellet waswashed and centrifuged. The supernatant was again aspirated, and thepellets were dissolved in IN NaOII and transferred to tubes for gammacounting. The data were used for Scatchard analysis (Munson and Rodband,Anal. Biochem. 107:220-239 (1980)) using the program Ligand (McPherson,Comput. Programs Biomed. 17: 107-114 (1983)). The results, shown inTable 9, indicate that humanized 2H7 variants had similar CD20 bindingaffinity as compared to murine 2H7, and similar binding affinity toRituxan®. It is expected that 2H7.v31 will have very similar K_(d) tov.16 on the basis of the binding shown in Table 7 above.

TABLE 9 Equilibrium binding affinity of 2H7 variants from Scatchardanalysis Antibody variant K_(d) (nM) n Rituxan 0.99 ± 0.49 3 2H7(murine) 1.23 ± 0.29 3 2H7.v16 0.84 ± 0.37 4 2H7.v73 1.22 ± 0.39 42H7.v75 1.09 ± 0.17 4

Example 8 Complement Dependent Cytotoxicity (CDC) Assays

2H7 IgG variants were assayed for their ability to mediatecomplement-dependent lysis of WIL2-S cells, a CD20 expressinglymphoblastoid B-cell line, essentially as described (Idusogie et al.,J. Immunol. 164:4178-4184 (2000); Idusogie et al., J. Immunol.166:2571-2575 (2001)). Antibodies were serially diluted 1:3 from a 0.1mg/mL stock solution. A 0.05 mL aliquot of each dilution was added to a96-well tissue culture plate that contained 0.05 ml of a solution ofnormal human complement (Quidel, San Diego, Calif.) To this mixture,50,000 W1L2-S cells were added in a 0.05 ml, volume. After incubationfor 2 h at 37° C., 0.05 ml, of a solution of Alamar blue (AccumedInternational, Westlake, Ohio) was added, and incubation was continuedfor an additional 18 h at 37° C. Covers were then removed from theplates, and they were shaken for 15 min at room temperature on anorbital shaker. Relative fluorescent units (RFU) were read using a 530nm excitation filter and a 590 nm emission filter. An EC₅₀ wascalculated by fitting RFU as a function of concentration for eachantibody using KaleidaGraph software.

The results (Table 10) show surprising improvement in CDC by humanized2H7 antibodies, with relative potency similar to Rituxan® for v.73,3-fold more potent than Rituxan® for v.75, and 3-fold weaker thanRituxan® for v.16.

TABLE 10 CDC activity of 2H7 antibodies compared to Rituxan. Numbers >1indicate less potent CDC activity than Rituxan ® and numbers <1 indicatemore potent activity than Rituxan ®. Antibody variant nEC₅₀(variant)/EC₅₀(Rituxan) Rituxan ® 4 -1-   2H7.v16 4 3.72; 4.082H7.v31* 4 2.21 2H7.v73 4 1.05 2H7.v75 4 0.33 2H7.v96* 4  0.9562H7.v114* 4  0.378 2H7.v115* 4  0.475 2H7.v116* 1 >100     2H7.v135* 20.42 Antibodies were produced from stable CHO lines, except that thoseindicated by * were produced transiently.

Example 9 Antibody Dependent Cellular Cytotoxicity (ADCC) Assays

2H7 IgG variants were assayed for their ability to mediateNatural-Killer cell (NK cell) lysis of WIL2-S cells, a CD20 expressinglymphoblastoid B-cell line, essentially as described (Shields et al., J.Biol Chem. 276:6591-6604 (2001)) using a lactate dehydrogenase (LDH)readout. NK cells were prepared from 100 mL of heparinized blood,diluted with 100 mL of PBS (phosphate buffered saline), obtained fromnormal human donors who had been isotyped for FcγRIII, also known asCD16 (Koene et al., Blood 90:1109-1114 (1997)). In this experiment, theNK cells were from human donors heterozygous for CD16 (F158/V158). Thediluted blood was layered over 15 mL of lymphocyte separation medium(ICN Biochemical, Aurora, Ohio) and centrifuged for 20 min at 2000 RPM.White cells at the interface between layers were dispensed to 4 clean50-mL tubes, which were filled with RPMI medium containing 15% fetalcalf serum. Tubes were centrifuged for 5 min at 1400 RPM and thesupernatant discarded. Pellets were resuspended in MACS buffer (0.5%BSA, 2 mM EDTA), and NK cells were purified using beads (NK CellIsolation Kit, 130-046-502) according to the manufacturer's protocol(Miltenyi Biotech,). NK cells were diluted in MACS buffer to 2×10⁶cells/mL.

Serial dilutions of antibody (0.05 ml.) in assay medium (F12/DMEM 50:50without glycine, 1 mM HEPES buffer pH 7.2, Penicillin/Streptomycin (100units/mL; Gibco), glutamine, and 1% heat-inactivated fetal bovine serum)were added to a 96-well round-bottom tissue culture plate. WIL2-S cellswere diluted in assay buffer to a concentration of 4×10⁵/mL. WIL2-Scells (0.05 mL per well) were mixed with diluted antibody in the 96-wellplate and incubated for 30 min at room temperature to allow binding ofantibody to CD20 (opsonization).

The ADCC: reaction was initiated by adding 0.1 mL of NK cells to eachwell. In control wells, 2% Triton X-100 was added. The plate was thenincubated for 4b at 37° C., Levels of LDH released were measured using acytotoxicity (LDH) detection kit (Kit #1644793, Roche Diagnostics,Indianapolis, Ind.) following the manufacturers instructions, 0.1 mL ofLDH developer was added to each well, followed by mixing for 10 s. Theplate was then covered with aluminum foil and incubated in the dark atroom temperature for 15 min. Optical density at 490 nm was then read anduse to calculate % lysis by dividing by the total LDH measured incontrol wells. Lysis was plotted as a function of antibodyconcentration, and a 4-parameter curve fit (KaleidaGraph) was used todetermine EC₅₀ concentrations.

The results showed that humanized 2H7 antibodies were active in ADCC,with relative potency 20-fold higher than Rituxan® for v.31 and v.75,5-fold more potent than Rituxan® for v.16, and almost 4-fold higher thanRituxan® for v.73.

TABLE 11 ADCC activity of 2H7 antibodies on WIL2-S cells compared to2H7.v16, based on n experiments. (Values >1 indicate lower potency than2H7.v16, and values <1 indicate greater potency.) Antibody variant nEC₅₀(variant)/EC₅₀(2H7.v16) Rituxan ® 4 5.3 2H7.v16 5 1 2H7.v31 1 0.242H7.v73 5 1.4 2H7.v75 4 0.25

Additional ADCC assays were carried out to compare combination-variantsof 2H7 with Rituxan®. The results of these assays indicated that2H7.v114 and 2H7.v115 have >10-fold improved ADCC potency as compared toRituxan® (Table 12).

TABLE 12 ADCC activity of 2H7 antibodies on WIL2-S cells compared toRituxan ®, based on n experiments (Values >1 indicate lower potency thanRituxan ®, and values <1 indicate greater potency). Antibody variantEC50(variant)/EC50(Rituxan) Rituxan ® 2 -1-   2H7 v.16 2 0.52 2H7 v.96 20.58 2H7.v114 2  0.093 2H7.v115 2  0.083 2H7.v116 2 0.30

Example 10 In Vivo Effects of 2H7 Variants in a Pilot Study inCynomolgus Monkeys

2H7 variants, produced by transient transfection of CHO cells, weretested in normal male cynomolgus (Macaca fascicularis) monkeys in orderto evaluate their in vivo activities. Other anti-CD20 antibodies, suchas C218 (Rituxan®) have demonstrated an ability to deplete B-cells innormal primates (Reff et al., Blood 83: 435-445 (1994)).

In one study, humanized 2H7 variants were compared. In a parallel study,Rituxan® was also tested in cynomolgus monkeys. Four monkeys were usedin each of five dose groups: (1) vehicle, (2) 0.05 mg/kg hu2H7.v16, (3)10 mg/kg hu2H7.v16, (4) 0.05 mg/kg hu2H17.v31, and (5) 10 mg/kghu2H7.v31. Antibodies were administered intravenously at a concentrationof 0, 0.2, or 20 mg/mL, for a total of two doses, one on day 1 of thestudy, and another on day 8. The first day of dosing is designated day 1and the previous day is designated day −1; the first day of recovery(for 2 animals in each group) is designated as day 11. Blood sampleswere collected on days −19, −12, 1 (prior to dosing), and at 6 h, 24 h,and 72 h following the first dose. Additional samples were taken on day8 (prior to dosing), day 10 (prior to sacrifice of 2 animals/group), andon days 36 and 67 (for recovery animals).

Peripheral B-cell concentrations were determined by a FACS method thatcounted CD3-/CD40+ cells. The percent of CD3-CD40+B cells of totallymphocytes in monkey samples were obtained by the following gatingstrategy. The lymphocyte population was marked on the forwardscatter/side scatter scattergram to define Region 1 (R1). Using eventsin R1, fluorescence intensity dot plots were displayed for CD40 and CD3markers. Fluorescently labeled isotype controls were used to determinerespective cutoff points for CD40 and CD3 positivity.

The results indicated that both 2H7.v16 and 2H7.v31 were capable ofproducing full peripheral B-cell depletion at the 10 mg/kg dose andpartial peripheral B-cell depletion at the 0.05 mg/kg dose (FIG. 11).The time course and extent of B-cell depletion measured during the first72 h of dosing were similar for the two antibodies. Subsequent analysisof the recovery animals indicated that animals treated with 2H7.v31showed a prolonged depletion of B-cells as compared to those dosed with2H7.v16. In particular, recovery animals treated with 10 mg/kg 2H7.v16,B-cells showed substantial B-cell recovery at some time between samplingon Day 10 and on Day 36. However, for recovery animals treated with 10mg/kg 2H7.v31, B-cells did not show recovery until some time between Day36 and Day 67 (FIG. 11). This suggests a greater duration of fulldepletion by about one month for 2H7.v31 compared to 2H7.v16.

No toxicity was observed in the monkey study at low or high dose and thegross pathology was normal. In other studies, v16 was well tolerated upto the highest dose evaluated of (100 mg/kg×2=1200 mg/m²×2) followingi.v. administration of 2 doses given 2 weeks apart in these monkeys.

Data in Cynomolgus monkeys with 2H7.v16 versus Rituxan® suggests that a5-fold reduction in CDC activity does not adversely affect potency. Anantibody with potent ADCC activity but reduced CDC activity may havemore favorable safety profile with regard to first infusion reactionsthan one with greater CDC activity.

Example 11 Fucose Deficient 2H7 Variant Antibodies with EnhancedEffector Function

Normal CHO and HEK293 cells add fucose to IgG oligosaccharide to a highdegree (97-98%). IgG from sera are also highly fucosylated.

DP12, a dihydrofolate reductase minus (DHFR⁻) CHO cell line that isfucosylation competent, and Lec13, a cell line that is deficient inprotein fucosylation were used to produce antibodies for this study. TheCHO cell line Pro-ILc13.6a (Lec13), was obtained from Professor PamelaStanley of Albert Einstein College of Medicine of Yeshiva University.Parental lines are Pro- (proline auxotroph) and Gat- (glycine,adenosine, thymidine auxotroph). The CHO-DP12 cell line is a derivativeof the CHO-KI cell line (ATCC #CCL-61), which is dihydrofolate reductasedeficient, and has a reduced requirement for insulin. Cell lines weretransfected with cDNA using the Superfect method (Qiagen, Valencia,Calif.). Selection of the Lec13 cells expressing transfected antibodieswas performed using puromycin dihydrochloride (Calbiochem. San Diego,Calif.) at 10 μg/ml in growth medium containing: MEM Alpha Medium withL-glutamine, ribonucleosides and deoxyribonucleosides (GIBCO-BRL,Gaithersburg, Md.), supplemented with 10% inactivated FBS (GIBCO), 10 mMHEPES, and IX penicillin/streptomycin (GIBCO). The CHO cells weresimilarly selected in growth medium containing Ham's F12 without GHT:Low Glucose DMEM without Glycine with NaHCO₃ supplemented with 5% FBS(GIBCO), 10 mM HEPES, 2 mM L-glutamine, IX GHT (glycine, hypoxanthine,thymidine), and 1× penicillin/streptomycin.

Colonies formed within two to three weeks and were pooled for expansionand protein expression.

The cell pools were seeded initially at 3×10⁶ cells/10 cm plate forsmall batch protein expression. The cells were converted to serum-freemedia once they grew to 90-95% confluency and after 3-5 days cellsupernatants were collected and tested in an Fe IgG- and IntactIgG-ELISA to estimate protein expression levels. Lec13 and CHO cellswere seeded at approximately 8×10⁶ cells/15 cm plate one day prior toconverting to PS24 production medium, supplemented with 10 mg/I,recombinant human insulin and 1 mg/L trace elements.

Lec13 cells and DP12 cells remained in serum-free production medium for3-5 days. Supernatants were collected and clarified by centrifugation in150 ml conical tubes to remove cells and debris. The protease inhibitorsPMSF and aprotinin (Sigma, St. Louis, Mo.) were added and thesupernatants were concentrated 5-fold on stirred cells using MWCO30filters (Amicon. Beverly, Mass.) prior to immediate purification usingprotein G chromatography (Amersham Pharmacia Biotech, Piscataway,N.J.)). All proteins were buffer exchanged into phosphate-bufferedsaline (PBS) using Centripriep-30 concentrators (Amicon) and analyzed bySDS-polyacrylamide gel electrophoresis. Protein concentrations weredetermined using A280 and verified using amino acid compositionanalysis.

The CHO cells were transfected with vectors expressing humanized 2H7v16,2H7v.31 and selected as described. The 2H7v.16 antibody retains the wildtype Fc region while v.31 (see Example 5. Table 7 above) has an Fcregion wherein 3 amino acid changes were made (S298A. E333A, K334A)which results in higher affinity for the FcγRIIIa receptor (Shields etal. J. Biol. Chem. 276 (9):6591-6604 (2001)). Following transfection andselection, individual colonies of cells were isolated and evaluated forprotein expression level and the highest producers were subjected tomethotrexate selection to select for cells that had amplified theplasmid copy number and which therefore produced higher levels ofantibody. Cells were grown, transferred to serum free medium for aperiod of 7 days, then the medium was collected, loaded onto a protein Acolumn and the antibody was eluted using standard techniques. The finalconcentration of the antibody was determined using an Elisa thatmeasures intact antibody. All proteins were buffer exchanged intophosphate-buffered saline (PBS) using Centripriep-30 concentrators.(Amicon) and analyzed by SDS-polyacrylamide gel electrophoresis.

Matrix-Assisted Laser Desorption/Ionization Time-of-flight (MALDI-TOF)Mass Spectral Analysis of Asparagine-Linked Oligosaccharides: N-linkedoligosaccharides were released from recombinant glycoproteins using theprocedure of Papac et al., Glycobiology 8, 445-454 (1998). Briefly, thewells of a 96 well PVDF-lined microtitre plate (Millipore, Bedford,Mass.) were conditioned with 100 μl methanol that was drawn through thePDVF membranes by applying vacuum to the Millipore Multiscreen vacuummanifold. The conditioned PVDF membranes were washed with 3×250 μlwater. Between all wash steps the wells were drained completely byapplying gentle vacuum to the manifold. The membranes were washed withreduction and carboxymethylation buffer (RCM) consisting of 6 Mguanidine hydrochloride, 360 mM Tris, 2 mM EDTA, pH 8.6. Glycoproteinsamples (50 μg) were applied to individual wells, again drawn throughthe PVDF membranes by gentle vacuum and the wells were washed with 2×50μl of RCM buffer. The immobilized samples were reduced by adding 50 μlof a 0.1 M dithiothreitol (DTT) solution to each well and incubating themicrotitre plate at 3C for 1 br. 171T was removed by vacuum and thewells were washed 4×250 μl water. Cysteine residues werecarboxymethylated by the addition of 50 μl of a 0.1 M iodoacetic acid(IAA) solution which was freshly prepared in 1 M NaOH and diluted to 0.1M with RCM buffer. Carboxymethylation was accomplished by incubation for30 min in the dark at ambient temperature. Vacuum was applied to theplate to remove the IAA solution and the wells were washed with 4×250 μlpurified water. The PVDF membranes were blocked by the addition of 100μl of 1% PVP360 (polyvinylpyrrolidine 360,000 MW) (Sigma) solution andincubation for 1 hr at ambient temperature. The PVP-360 solution wasremoved by gentle vacuum and the wells were washed 4×250 μl water. ThePNGase F (New England Biolabs, Beverly, Mass.) digest solution, 25 μl ofa 25 Unit/mil solution in 10 mM Tris acetate, pH 8.4, was added to eachwell and the digest proceeded for 3 hr at 37° C. After digestion, thesamples were transferred to 500 μl Eppendorf tubes and 2.5 μL of a 1.5 Macetic acid solution was added to each sample. The acidified sampleswere incubated for 3 hr at ambient temperature to convert theoligosaccharides from glycosylamines to the hydroxyl form. Prior toMALDI-TOF mass spectral analysis, the released oligosaccharides weredesalted using a 0.7-ml bed of cation exchange resin (AG50W-X8 resin inthe hydrogen form) (Bio-Rad, Hercules. Calif.) slurried packed intocompact reaction tubes (US Biochemical, Cleveland, Ohio.).

For MALDI-TOF mass spectral analysis of the samples in the positivemode, the desalted oligosaccharides (0.5 μl aliquots) were applied tothe stainless target with 0.5 μl of the 2,5 dihydroxybenzoic acid matrix(sDHB) that was prepared by dissolving 2 mg 2,5 dihydroxybenzoic acidwith 0.1 mg of 5-methoxyslicylic acid in 1 ml of ethanol/10 mM sodiumchloride 1:1 (v/v). The sample/matrix mixture was dried by vacuum. Foranalysis in the negative mode, the desalted N-linked oligosaccharides(0.5 μl aliquots) were applied to the stainless target along with 0.5 μl2′,4′,6′-trihydroxyacetophenonc matrix (THAP) prepared in 1:3 (v/v)acetonitrile/13.3 mM ammonium citrate buffer. The sample/matrix mixturewas vacuum dried and then allowed to absorb atmospheric moisture priorto analysis. Released oligosaccharides were analyzed by MALDI-TOF on aPerSeptive BioSystems Voyager-DE mass spectrometer. The massspectrometer was operated at 20 kV either in the positive or negativemode with the linear configuration and utilizing delayed extraction.Data were acquired using a laser power of 1300 and in the data summationmode (240 scans) to improve the signal to noise. The instrument wascalibrated with a mixture of standard oligosaccharides and the data wassmoothed using a 19 point Savitsky-Golay algorithm before the masseswere assigned. Integration of the mass spectral data was achieved usingCaesar 7.0 data analysis software package (SciBridge Software).

Natural Killer (NK) Cell Antibody Dependent Cytoxicity Assays.

ADCC assays were performed as described in Example 9. NK to target cell(WIL2-S) ratio was 4 to 1, assays were run for 4 hours, and toxicity wasmeasured as before using lactose dehydrogenase assay. Target cells wereopsonized with the concentrations of antibody indicated for 30 min priorto addition of NK cells. The Rituxan® antibody used was from Genentech(S. San Francisco, Calif.). FIG. 12 shows the results of arepresentative ADCC assay.

The results show that underfucosylated antibodies mediate NK cell targetcell killing more efficiently than do antibodies with a full complementof fucose. The underfucosylated antibody, 2H7v.31, is most efficient atmediating target cell killing. This antibody is effective at lowerconcentrations and is capable of mediating killing of a greaterpercentage of target cells at higher concentrations than am the otherantibodies. The activity of the antibodies is as follows: Lec13-derived2H7 v31>Lec 13 derived 2H7v16>Dp12 derived 2H7v31>Dp 12 derived 2H7v16>or = to Rituxan. The protein and carbohydrate alterations are additive.Comparison of the carbohydrate found on native IgG from theLec13-produced and CHO-produced IgG showed no appreciable differences inthe extent of galactosylation and hence the results can be attributedsolely to the presence/absence of fucose.

Example 12 Fucose-Deficient 2H7 Variant Antibodies with Enhanced ADCC InVivo

This example describes ADCC activity in vivo of the fucose-deficienthumanized 2H7 variants including v.16 and v.31 produced in Lec13compared to normal fucosylated counterparts produced in DP12, in miceexpressing human CD16 [FcγRIII] and human CD20.

Generation of huCD20Tg⁺ huCD16Tg⁺ mCD16^(−/−) mice Human CD20 transgenicmice were generated from human CD2 BAC DNA (Invitrogen, Carlsbad,Calif.). Mice were screened based on the FACS analysis of human CD20expression. HuCD20 Tg⁺ mice were then crossed with huCD16Tg⁺mCD16^(−/−)mice to generate huCD20Tg⁺huCD16Tg⁺ mCD16^(−/−) mice.

In Vivo Treatment

Ten to 100 μg of each of the 2H7 variants or Rituxan® is administratedto huCD20Tg⁺ huCD16Tg⁺mCD16^(−/−) mice via intraperitoneal injections.Equal amount of isotype-matched antibodies will be applied similarly tothe negative control group of animals.

Mouse Lymphocytes Preparation

Mouse lymphocytes from whole blood, spleen, lymph nodes and bone marroware prepared according to standard protocol described in “CurrentProtocols in Immunology, edited by John Coligan, Ada Kruisbeek, DavidMargulies, Ethan Shevach and Warren Strober, 1994”.

FACS Analysis

Half million cells are washed and resuspended in 100 μl of FACS buffer,which is phosphate buffered saline with 1% BSA, containing 5 μl ofstaining or control antibody. All the staining antibodies, includingisotype controls, are obtained from PharMingen, San Diego, Calif. HumanCD20 expression is assessed by staining with Rituxan® along withFITC-conjugated anti-human IgG1 secondary antibody. FACS analysis isconducted using FACScan and Cell Quest (Becton Dickinson ImmunocytometrySystems, San Jose, Calif.). All the lymphocytes are defined in theforward and side light scatterings, while all the B lymphocytes aredefined with the expression of B220 on the cell surface.

B cell depletion and recovery are assessed by analyzing peripheral Bcell counts and analysis of hCD20+ B cells by FACS in the spleen, lymphnode and bone marrow on a daily basis for the first week after injectionand thereafter on a weekly basis. Serum levels of the injected 2H7variant antibody are monitored.

The results of this in vivo assay confirms the in vitro findings on theincreased ADCC activity and greater B cell depletion of fucose-deficient2H7 variants over wild-type (with respect to fucosylation) glycosylationcounterparts.

Example 13 Apoptosis Activity

Anti-CD20 antibodies including Rituxan® have been shown to induceapoptasis in vitro when crosslinked by a secondary antibody or bychemical means (Shan et al., Blood 9:1644-1652 (1998); Byrd et al.,Blood 99:103843 (2002); Pederson et al., Blood 99:1314-19 (2002)). Whenchemically crosslinked, murine 2H7 dimers induced apoptosis of Daudicells (Ghetie et al., Pro Natl Acad Sci USA 94:7509-14 (1997)).Crosslinking with a secondary antibody also induced apoptosis with themurine 2H7 antibody (Shan et al., 1998). These activities are believedto be physiologically relevant because a variety of mechanisms couldlead to crosslinking of anti-CD20 antibodies bound to cell-surface CD20in vivo.

RhuMAb 2H7.v16 [humanized 2H7 v16; RhuMAb stands for recombinant humanmonoclonal antibody] and Rituxan® were compared in apoptosis assays invitro using a secondary crosslinking antibody. Ramos cells (CRL-1596,ATCC, Manassas, Va.), a CD20-expressing, human B lymphocyte cell line,were used to measure the ability of the anti-CD20 monoclonal antibodiesrhuMAb 2H7.v16 and Rituximab versus a negative-control antibody.Trastuzumab (Herceptin®. Genentech, South San Francisco. Calif.), toinduce apoptosis as measured through Annexin V staining and propidiumiodide dye exclusion (Vybrant® Apoptosis Assay Kit, Molecular Probes,Seattle, Wash.). The Ramos cells were cultured in RPMI-1640 medium(Gibco, Rockville, Md.) containing 10% fetal bovine serum (BiosourceInternational, Camarillo, Calif.) and 2 mM L-glutamine (Gibco). Prior tobeing assayed, the cells were washed twice in fresh media and thenadjusted to a cell concentration of 2×10⁶ per mL. Cells (150 μL) wereadded to 96-well assay plates (Becton Dickinson, Palo Alto, Calif.)which contained 150 μL of a predetermined amount of control IgG1, rhuMAb2H7.v16, or Rituximab, along with F(ab)′2 goat anti-human Fc (PierceBiotechnology, Rockford, Ill.). The final IgG concentrations were 100,10, 1.0, 0.1, 0.01 and 0.001 nM, and the F(ab)′2 goat anti-human Fcantibody concentration was set at twice the respective sample antibodyconcentration. Each dilution was set up in triplicate. After a 24-hourincubation at 37° C., the cells were washed twice with PBS and thenstained with Annexin V and propidium iodide according to themanufacturer's recommendations. The staining patterns of the Ramos cellswere analyzed by flow cytometry using a FACscan Flow Cytometer (BectonDickinson, San Jose, Calif.), and data were collected for 10 s-periods.The data were reduced using the Cellquest Pro software (BectonDickinson). Ramos cells that were positive for (1) Annexin V staining,(2) Annexin V and propiduim iodide double-staining, and (3) the numberof unstained live cells, were counted and plotted using KaleidaGraphsoftware (Synergy Software, Reading, Pa.).

Both rhuMAb 2H7.v16 and Rituximab induced apoptosis of Ramos cells whencrosslinked with anti-human Fc and as compared to an irrelevant IgG1control antibody (FIGS. 13-15). The apoptotic activity of (rhuMAb 2H7)was slightly lower than that of Rituximab. At 10 nM concentrations ofcrosslinked rhuMAb 2H7, Rituximab, and control IgG1 antibody, fractionsof Annexin V stained cells were 18.5, 16.5, 2.5%, respectively,fractions of doubly labeled cells were 29, 38, and 16%, and numbers oflive cells counted per 10 s were 5200, 3100, and 8600.

These in vitro data demonstrate that apoptosis is one potentialmechanism for in vivo B cell depletion. In vivo crosslinking of rhuMAb2H7 or Rituximab bound to cell-surface CD20 may occur through FcγR onthe surfaces of immune effector cells.

Example 14 In Vivo Suppression of Tumor Growth

The ability of rhuMAb 2H7.v16 to inhibit the growth of the Raji humanB-cells, a lymphoma cell line (ATCC CCL 86), was evaluated in Balb/cnude (athymic) mice. The Raji cells express CD20 and have been reportedto grow in nude mice, producing metastatic disease; tumor growth isinhibited by Rituxan®(Clynes et al., Nature Medicine 6, 443-446 (2000)).Fifty-six 8-10 week old, Balb/c nude mice were divided into 7 groups(A-G) with each group consisting of 8 mice. On day 0, each mousereceived a subcutaneous injection of 5×10⁶ Raji B-lymphoma cells in theflank. Beginning at day 0, each mouse received either 100 uL of thenegative-control solution (PBS; phosphate-buffered saline). Rituxan® or2H7.v16. Dosage was dependent on weight and drug delivery wasintravenously via the tail vein. Group A mice received PBS. Groups B-Dreceived Rituxan® at 5.0, mg/kg, 0.5 mg/kg, and 0.05 mg/kg respectively.Groups E-G mice received 2H7 v.16 at 5.0 mg/kg, 0.5 mg/kg, and 0.05mg/kg respectively. The injections were repeated every week for 6 weeks.At weekly intervals during treatment, each mouse was inspected for thepresence of palpable tumors at the site of injection, and the volume ofthe tumors if present were measured and recorded. A final inspection wasmade at week 8 (after a two-week interval of no treatments).

The results of this study showed that both rhuMAb 2H7.v16 and Rituxan®and were effective at inhibiting subcutaneous Raji-cell tumor growth innude mice (FIGS. 16-18). Tumor growth was observed in the PBS controlgroup beginning at 4 weeks. However, no tumor growth was observed ingroups treated with Rituxan® or 2H7.v16 at 5 mg/kg or 0.5 mg/kg for the8-week duration of the study. In the low-dose 0.05 mg/kg treatmentgroups, tumors were observed in one animal in the 2H7 group and in oneanimal in the Rituxan® group (FIG. 1g ).

Example 15 Cloning of Cynomolgus Monkey CD20 and Antibody Binding

The CD20 DNA sequence for cynomolgus monkey (Macaca fascicularis) wasdetermined upon the isolation of cDNA encoding CD20 from a cynomolgusspleen cDNA library. A SUPERSCRIPT™ Plasmid System for cDNA Synthesisand Plasmid Cloning (Cat #I18248-013, Invitrogen, Carlsbad, Calif.) wasused with slight modifications to construct the library. The cDNAlibrary was ligated into a pRK5E vector using restriction sites Xho I-and Not 1. mRNA was isolated from spleen tissue ((California RegionalResearch Primate Center, Davis, Calif.). Primers to amplify cDNAencoding CD20 were designed based on non-coding sequences of human CD20.N-terminal region primer 5′-AGTTTTGAGAGCAAAATG-3′ (SEQ ID NO. 37) andC-terminal region primer 5′-AAGCTATGAACACTAATG-3′ (SEQ ID NO. 38) wereused to clone by polymerase chain reaction (PCR) the cDNA encodingcynomolgus monkey CD20. The PCR reaction was carried out using PlatinumTaq DNA Polymerase High Fidelity according to the manufacturersrecommendation (Gibco, Rockville, Md.). The PCR product was subclonedinto pCR®2,1-TOPO® Vector (Invitrogen) and transformed into XL-1 blue E.coli (Stratagene. La Jolla, Calif.). Plasmid DNA containing ligated PCRproducts was isolated from individual clones and sequenced.

The amino acid sequence for cynomolgus monkey CD20 is shown in FIG. 19.FIG. 20 shows a comparison of cynomolgus and human CD20. The cynomolgusmonkey CD20 is 97.3% similar to human CD20 with 8 differences. Theextracellular domain contains one change at V157A, while the remaining 7residues can be found in the cytoplasmic or transmembrane regions.

Antibodies directed against human CD20 were assayed for the ability tobind and displace FITC-conjugated murine 2H7 binding to cynomolgusmonkey cells expressing CD20. Twenty milliliters of blood were drawnfrom 2 cynomolgus monkeys (California Regional Research Primate Center,Davis, Calif.) into sodium heparin and shipped directly to GenentechInc., On the same day, the blood samples were pooled and diluted 1:1 bythe addition of 40 ml of phosphate buffered saline (PBS). 20 ml ofdiluted blood was layered on 4×20 ml of Ficoll-Paque™ Plus (AmershamBiosciences, Uppsala, Sweden) in 50 ml conical tubes (Cat #352098,Falcon, Franklin Lakes. N.J.) and centrifuged at 1300 rpm for 30 minutesR.T, in a Sorval 7 centrifuge. (Dupont, Newtown, Conn.). The PBMC layerwas isolated and washed in PBS. Red blood cells were lysed in a 0.2%NaCl solution, restored to isotonicity with an equivalent volume of a1.6% NaCl solution, and centrifuged for 10 minutes at 1000 RPM. The PBMCpellet was resuspended in RPMI 1640 (Gibco, Rockville, Md.) containing5% fetal bovine serum (FBS) and dispensed into a 10 cm tissue culturedish for 1 hour at 37° C. The non-adherent B and T cell populations wereremoved by aspiration, centrifuged and counted. A total of 2.4×10⁷ cellswere recovered. The resuspended PBMC were distributed into twenty 12×75mm culture tubes (Cat #352053, Falcon), with each tube containing 1×10⁶cells in a volume of 0.25 ml. Tubes were divided into four sets of fivetubes. To each set was added either media (RPMI1640, 5% FBS), titratedamounts of control human IgG, antibody, Rituxan, 2H7.v16, or 2H7.v31,The final concentration of each antibody was 30, 10, 3.3 and 1.1 nM. Inaddition, each tube also received 20 ul of Fluorescein Isothiocyanate(FITC)-conjugated anti-human CD20 (Cat #555622, BD Biosciences, SanDiego, Calif.). The cells were gently mixed, incubated for 1 hour on iceand then washed twice in cold PBS. The cell surface staining wasanalyzed on a Epic XL-MCL (Coulter, Miami. Fla.), the geometric meansderived, plotted (KaleidaGraph™, Synergy Software, Reading, Pa.) versusantibody concentrations.

Data in FIG. 21 showed that 2H7 v.16 and 2H7 v.31 competitivelydisplaced FITC-murine 2H17 binding to cynomolgus monkey cells.Furthermore, Rituxan® also displaced FITC-murine 2H7 binding thusdemonstrating that both 2H17 and Rituxan® bind to an overlapping epitopeon CD20. In addition, the data show that the IC₅₀ value for 2H7 v.16,2H7 v.31 and Rituxan am similar and fall in the 4-6 nM range.

Example 16 Phase I/II Study of rhuMAb 2H7 (2H7.v16) in Moderate toSevere Rheumatoid Arthritis Protocol Synopsis

A randomized, placebo-controlled, multicenter, blinded phase I/II studyof the safety of escalating doses of PRO70769 (rhuMAb 2H7) in subjectswith moderate to severe rheumatoid arthritis receiving stable doses ofconcomitant methotrexate.

Objectives

The primary objective of this study is to evaluate the safety andtolerability of escalating intravenous (IV) doses of PR070769 (rhuMAb2H7) in subjects with moderate to severe rheumatoid arthritis (RA).

Study Design

This is a randomized, placebo-controlled, multicenter, blinded PhaseI/II, investigator- and subject-blinded study of the safety ofescalating doses of PRO70769 in combination with MTX in subjects withmoderate to severe RA. The study consists of a dose escalation phase anda second phase with enrollment of a larger number of subjects. TheSponsor will remain unblinded to treatment assignment.

Subjects with moderate to severe RA who have failed one to fivedisease-modifying antirheumatic drugs or biologics who currently haveunsatisfactory clinical responses to treatment with MTX will beenrolled.

Subjects will be required to receive MTX in the range of 10-25 mg weeklyfor at least 12 weeks prior to study entry and to be on a stable dosefor at least 4 weeks before receiving their initial dose of study drug(PRO70769 or placebo). Subjects may also receive stable doses of oralcorticosteroids (up to 10 mg daily or prednisone equivalent) and stabledoses of nonsteroidal anti-inflammatory drugs (NSAIDs). Subjects willreceive two IV infusions of PRO70769 or placebo equivalent at theindicated dose on Days 1 and 15 according to the following doseescalation plan (see FIG. 22).

Dose escalation will occur according to specific criteria and afterreview of safety data by an internal safety data review committee andassessment of acute toxicity 72 hours following the second infusion inthe last subject treated in each cohort. After the dose escalationphase, 40 additional subjects (32 active and 8 placebo) will berandomized to each of the following dose levels: 2×50 mg, 2×200 mg,2×500 mg, and 2×1000 mg, if the dose levels have been demonstrated to betolerable during the dose escalation phase. Approximately 205 subjectswill be enrolled in the study.

B-cell counts will be obtained and recorded. B-cell counts will beevaluated using flow cytometry in a 48-week follow-up period beyond the6-month efficacy evaluation. B-cell depletion will not be considered adose-limiting toxicity (DLC), but rather the expected pharmacodynamicoutcome of PRO70769 treatment.

In an optional substudy, blood for serum and RNA analyses, as well asurine samples will be obtained from subjects at various timepoints.These samples may be used to identify biomarkers that may be predictiveof response to PRO70769 treatment in subjects with moderate to severeRA.

Outcome Measures

The primary outcome measure for this study is the safety andtolerability of PRO70769 in subjects with moderate to severe RA.

Study Treatment

Cohorts of subjects will receive two IV infusions of PRO70769 or placeboequivalent at the indicated dose on Days 1 and 15 according to thefollowing escalation plan:

-   -   mg PRO70769 or placebo equivalent: 4 subjects active drug, 1        control    -   50 mg PRO70769 or placebo equivalent: 8 subjects active drug, 2        control    -   200 mg PRO70769 or placebo equivalent: 8 subjects active drug, 2        control    -   500 mg PRO70769 or placebo equivalent: 8 subjects active drug, 2        control    -   1000 mg PRO70769 or placebo equivalent: 8 subjects active drug,        2 control

Efficacy

The efficacy of PRO70769 will be measured by ACR responses. Thepercentage of subjects who achieve an ACR20, ACR50, and ACR70 responsewill be summarized by treatment group and 95% confidence intervals willbe generated for each group. The components of these response and theirchange from baseline will be summarized by treatment and visit.

CONCLUSION

The data above demonstrated the success in producing humanized CD20binding antibodies, in particular humanized 2H7 antibody variants, thatmaintained and even enhanced their biological properties. The humanized2H7 antibodies of the invention bound to CD20 at affinities similar tothe murine donor and chimeric 21H7 antibodies and were effective at Bcell killing in a primate, leading to B cell depletion. Certain variantsshowed enhanced ADCC over a chimeric anti-CD20 antibody currently usedto treat NHL, favoring the use of lower doses of the therapeuticantibody in patients. Additional, whereas it may be necessary for achimeric antibody that has murine FR residues to be administered at adose effective to achieve complete B cell depletion to obviate anantibody response against it, the present humanized antibodies can beadministered at dosages that achieve partial or complete B celldepletion, and for different durations of time, as desired for theparticular disease and patient. In addition, these antibodiesdemonstrated stability in solution. These properties of the humanized2H7 antibodies make them ideal for use as immunotherapeutic agent in thetreatment of CD20 positive cancers and autoimmune diseases; theseantibodies are not expected to be immunogenic or will at least be lessimmunogenic than fully murine or chimeric anti-CD20 antibodies in humanpatients.

REFERENCES

References cited within this application, including patents, publishedapplications and other publications, are hereby incorporated byreference.

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of molecular biology and the like,which are within the skill of the art. Such techniques are explainedfully in the literature. See e.g., Molecular Cloning: A LaboratoryManual, (J. Sambrook et al., Cold Spring Harbor Laboratory. Cold SpringHarbor, N.Y., 1989); Current Protocols in Molecular Biology (F. Ausubelet al., eds., 1987 updated); Essential Molecular Biology(T. Brown ed.,IRL Press 1991); Gene Expression Technology (Goeddel ed., Academic Press1991); Methods for Cloning and Analysis of Eukaryotic Genes (A. Bothwellet al., eds., Bartlett Publ. 1990); Gene Transfer and Expression (M.Kriegler, Stockton Press 1990); Recombinant DNA Methodology II (R. Wu etal., eds., Academic Press 1995); PCR: A Practical Approach (M. McPhersonet al., IRL Press at Oxford University Press 1991); OligonucleotideSynthesis (M. Gait ed., 1984); Cell Culture for Biochemists (R. Adamsed., Elsevier Science Publishers 1990); Gene Transfer Vectors forMammalian Cells (J. Miller & M. Calos eds., 1987); Mammalian CellBiotechnology (M. Butler ed., 1991); Animal Cell Culture (J. Pollard etal., eds., Humana Press 1990); Culture of Animal Cells, 2^(nd) Ed. (R.Freshney et al., eds., Alan R. Liss 1987); Flow Cytometry and Sorting(M. Melamed er al, eds., Wiley-Liss 1990); the series Methods inEnzymology (Academic Press, Inc.); Wirth M. and Hauser H. (1993);Immunochemistry in Practice, 3rd edition, A. Johnstone & R. Thorpe,Blackwell Science, Cambridge, Mass., 1996; Techniques inImmunocytochemistry (G. Bullock & P. Petrusz eds., Academic Press 1982,1983, 1985, 1989); Handbook of Experimental Immunology, (D. Weir & C.Blackwell, eds.); Current Protocols in Immunology (J. Coligan et al,eds. 1991); Immunoassay (E. P. Diamandis & T. K. Christopoulos, eds.,Academic Press, Inc., 1996); Goding (1986) Monoclonal Antibodies:Principls and Practice (2d ed) Academic Press, New York; Ed Harlow andDavid Lane, Antibodies A laboraty Manual. Cold Spring Harbor Laboratory,Cold Spring Harbor. N.Y., 1988; Antibody Engineering, 2 edition (C.Borrebaeck, ed., Oxford University Press, 1995); and the series AnnualReview of Immunology; the series Advances in Immunology.

1-81. (canceled) 82: An isolated nucleic acid encoding an antibody thatbinds to human CD20 or an antigen-binding fragment thereof, wherein theantibody heavy chain variable region comprises the amino acid sequenceof SEQ ID NO:8 with amino acid substitutions D56A and N100A and theantibody light chain variable region comprises the amino acid sequenceof SEQ ID NO:2 with amino acid substitution S92A. 83: A vectorcomprising the nucleic acid of claim
 82. 84: An isolated host cellcomprising the nucleic acid of claim
 82. 85: An isolated host cellcomprising the vector of claim
 83. 86: An isolated host cell comprisinga first vector comprising a nucleic acid encoding the heavy chainvariable region (VH) of an antibody or antigen-binding fragment thereof,and a second vector comprising a nucleic acid encoding the light chainvariable region (VL) of the antibody or antigen-binding fragmentthereof, wherein the antibody heavy chain variable region comprises theamino acid sequence of SEQ ID NO:8 with amino acid substitutions D56Aand N100A and the antibody light chain variable region comprises theamino acid sequence of SEQ ID NO:2 with amino acid substitution S92A;and wherein the antibody binds to human CD20. 87: A method of producingan antibody that binds to human CD20 or an antigen-binding fragmentthereof, encoded by the nucleic acid present in the host cell of claim84, comprising culturing the host cell under suitable conditions so thatthe antibody or antigen-binding fragment thereof is produced. 88: Amethod of producing an antibody that binds to human CD20 or anantigen-binding fragment thereof, encoded by the nucleic acid in thevector present in the host cell of claim 85, comprising culturing thehost cell under suitable conditions so that the antibody orantigen-binding fragment thereof is produced. 89: A method of producingan antibody that binds to human CD20 or antigen-binding fragmentthereof, comprising culturing a host cell under conditions so that theantibody or antigen-binding fragment is produced, wherein the host cellcomprises a first vector and a second vector encoding the antibody orantigen-binding fragment thereof, wherein the first vector comprises anucleic acid encoding the heavy chain variable region (VH) of theantibody, and the second vector comprises a nucleic acid encoding thelight chain variable region (VL) of the antibody, wherein the antibodyheavy chain variable region comprises the amino acid sequence of SEQ IDNO:8 with amino acid substitutions D56A and N100A and the antibody lightchain variable region comprises the amino acid sequence of SEQ ID NO:2with amino acid substitution S92A. 90: An isolated nucleic acid encodingan antibody that binds to human CD20 or an antigen-binding fragmentthereof, wherein the antibody heavy chain variable region comprises theamino acid sequence of SEQ ID NO:8 with amino acid substitutions D56Aand N100A and the antibody light chain variable region comprises theamino acid sequence of SEQ ID NO:2 with amino acid substitutions S26A,S56A and S92A. 91: A vector comprising the nucleic acid of claim
 90. 92:An isolated host cell comprising the nucleic acid of claim
 90. 93: Anisolated host cell comprising the vector of claim
 91. 94: A method ofproducing an antibody that binds to human CD20 or an antigen-bindingfragment thereof, encoded by the nucleic acid present in the host cellof claim 92, comprising culturing the host cell under suitableconditions so that the antibody or antigen-binding fragment thereof isproduced. 95: A method of producing an antibody that binds to human CD20or an antigen-binding fragment thereof, encoded by the nucleic acid inthe vector present in the host cell of claim 93, comprising culturingthe host cell under suitable conditions so that the antibody orantigen-binding fragment thereof is produced. 96: A method of producingan antibody that binds to human CD20 or antigen-binding fragmentthereof, comprising culturing a host cell under conditions so that theantibody or antigen-binding fragment is produced, wherein the host cellcomprises a first vector and a second vector encoding the antibody orantigen-binding fragment thereof, wherein the first vector comprises anucleic acid encoding the heavy chain variable region (VH) of theantibody, and the second vector comprises a nucleic acid encoding thelight chain variable region (VL) of the antibody, wherein the antibodyheavy chain variable region comprises the amino acid sequence of SEQ IDNO:8 with amino acid substitutions D56A and N100A and the antibody lightchain variable region comprises the amino acid sequence of SEQ ID NO:2with amino acid substitutions S26A, S56A and S92A.